393 Estrogen receptor a-induced cholecystokinin type A receptor expression in the female mouse pituitary Hyun Joon Kim1,2, Mary C Gieske1,3, Susan Hudgins1, Beob Gyun Kim4, Andree Krust5, Pierre Chambon5 and CheMyong Ko1,3 1Division of Clinical and Reproductive Sciences, Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky 40536, USA 2Department of Anatomy and Neurobiology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, South Korea Departments of 3Biology and 4Animal Sciences, University of Kentucky, Lexington, Kentucky 40536, USA 5Institut de Genetique et de Biologie Moleculaire et Cellulaire (CNRS, INSERM, ULP, College de France) and Institut Clinique de la Souris, BP10142, 67404 Illkirch-Strasbourg, France (Correspondence should be addressed to C Ko; Email: [email protected]) Abstract Estrogen plays a critical role in inducing LH surge. In the seen in cultured primary anterior pituitary cells, indicating pituitary, estrogen receptor a (ERa) mediates the action of that estrogen directly acts on pituitary cells to induce CCK- estrogen, while the downstream pathway of ERa activation is AR expression. Immunohistological analysis revealed that yet to be elucidated. Here, we report the finding that more than 80% of gonadotrophs express CCK-AR in the cholecystokinin type A receptor (CCK-AR) is an ERa afternoon of proestrus. To test whether CCK-AR mediated downstream gene in the mouse anterior pituitary. In the the sensitizing effect of estrogen in GnRH-induced LH cycling mouse pituitary, the expression of CCK-AR mRNA secretion, primary pituitary cells were primed with estrogen is markedly higher in the afternoon of proestrus compared followed by treatment with GnRH in the presence or absence with metestrus. Both ovariectomy (OVX) and null mutation of lorglumide, a CCK-AR antagonist. While both groups of the ERa gene completely abolish CCK-AR mRNA secreted LH upon GnRH treatment, lorglumide treatment expression. Injection of 17b-estradiol to OVX wild-type significantly decreased LH secretion. Taken together, this mice induces recovery of CCK-AR mRNA expression to study finds CCK-AR to be an ERa downstream gene in the levels observed at proestrus, but no such recovery is induced pituitary and suggests that CCK-AR may play a role in the in OVX ERa knockout mice. The same pattern of estrogen estrogen sensitization of the pituitary response to GnRH. dependency in inducing CCK-AR mRNA expression was Journal of Endocrinology (2007) 195, 393–405 Introduction As a nuclear receptor transcription factor, ERa has been speculated to regulate the expression of molecules involved in It is well established that, among multiple factors that hormone secretion in the gonadotrophs (Naik et al.1985, contribute to the induction of luteinizing hormone (LH) Powers 1986, Sapino et al.1986, Bauer-Dantoin et al.1993, secretion, the ovarian steroid estrogen plays a pivotal role Thomas & Clarke 1997, Kirkpatrick et al.1998, DePasquale by exerting positive feedback to the pituitary (Clarke 2002, 1999, Clarke 2002, Rispoli & Nett 2005). In this regard, it is Christian et al. 2005); however, the process by which interesting that the cholecystokinin (CCK)/CCK type A estrogen controls these events has not been fully under- receptor (CCK-AR) system, a well-known regulatory stood. Estrogen plays its role by modulating the activity of machinery of protein secretion, has been detected in the a and/or b subtypes of estrogen receptors (ERs) in a tissue pituitary and shown to be involved in the LH secretion (Vijayan type-dependent manner. While both ERa and ERb are et al.1979, Vijayan & McCann 1986, Peuranen et al.1995). present in the pituitary, ERa has been shown to be the Furthermore, recently, it has been shown that estrogen via ERa effector of estrogen action in the pituitary (Sanchez-Criado influences the function and expression of the CCK/CCK-AR et al. 2004, 2005). In support of this finding, ERa system in regulating satiety (Geary et al.1994, 1996, 2001). knockout (ERaKO) female mice are completely infertile These findings have led us to hypothesize that as a way of and do not ovulate, while ERbKO mice are fertile regulating LH secretion, estrogen via ERa may modulate (Dupont et al. 2000, Hewitt & Korach 2003). However, CCK/CCK-AR expression in the pituitary. the downstream pathway of ERa activation in the pituitary CCK is a multifunctional peptide, whose action is mediated gonadotroph is not yet known. by two forms of G-protein-coupled receptors, CCK-AR and Journal of Endocrinology (2007) 195, 393–405 DOI: 10.1677/JOE-07-0358 0022–0795/07/0195–393 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/30/2021 06:58:45AM via free access 394 HJKIMand others . CCK-AR in the female mouse pituitary type B (CCK-BR; Wank 1995, Williams et al.2002). CCK perfusion was performed using 4% neutralized buffered stimulates the secretion of a variety of proteins including paraformaldehyde. After postfixation with the same fixative, digestive enzymes (Sankaran et al.1980, Rossetti et al.1987), tissues were stored in 20% sucrose and later frozen in OCT neuropeptides (Wank 1995, Tirassa et al.1998), and hormones compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA). (Rossetti et al.1987, Karlsson & Ahren 1992, Peuranen et al. For determination of stages of the estrous cycle, a standard 1995, Andren-Sandberg et al.1999). Since the first cloning of vaginal lavage technique (Becker et al.2005) was applied. After CCK-AR in the pancreatic acinar cells (Sankaran et al.1980), daily vaginal lavages for 2 weeks, mice were killed on proestrus expression of CCK-AR has been reported in multiple cell types or metestrus at 1500 h to collect estrus cycle-specific pituitary including gastric chief cells (Qian et al.1993), smooth muscle tissues. For primary pituitary culture, 10-week-old female mice cells of gastrointestinal tract (Bitar & Makhlouf 1982, Meyer (C57BL/6) were purchased from Harlan Animal Breeding et al.1989), neurons (Skirboll et al.1986), and endocrine cells Center (Harlan, Indianapolis, IN, USA). (Kamilaris et al.1992). In particular, the CCK/CCK-AR system has been implicated in the secretion of pituitary Generation of ERaKO mice hormones including adrenocorticotropic hormone, K K b-endorphin, growth hormone (GH), thyroid-stimulating The generation of ERaKO (ERa / ) mice resulted from a hormone (TSH), prolactin (PRL), and LH (Vijayan et al. cross of male ERaflox/flox with female Zp3cre, a line expressing 1979, Vijayan & McCann 1986, Bondy et al.1989, Kamilaris Cre recombinase specifically in the oocyte. ERaflox/flox mice et al.1992, Mannisto et al.1992, Peuranen et al.1995). possess two loxP sites flanking exon 3 of the ERa gene Here, we report evidence that estrogen induces CCK-AR (Dupont et al.2000). The resulting F1 heterozygote C expression via ERa in the pituitary and CCK-AR activation ERaflox/ Zp3cre was then bred with ERaflox/flox to produce enhances sensitivity of estrogen-primed gonadotrophs to ERaflox/flox Zp3cre. Female ERaflox/flox Zp3cre mice produce K gonadotropin-releasing hormone (GnRH)-stimuli. ERa oocytes due to the deletion of floxed exon in the oocyte. Thus, oocytes fertilized by sperm from ERaflox/flox K males result in progeny that are ERaflox/ . The breeding of K female ERaflox/flox Zp3cre with male ERaflox/ mice Materials and Methods K K produces half of progeny that are ERa / . Genotyping was performed by PCR using ear biopsy DNA. Genomic Reagents DNA was isolated from ear using the Easy-DNA Kit Antibodies for CCK-AR were purchased from Santa Cruz (Invitrogen). A primer set of ERaP1 (50-ttg ccc gat aac aat Biotechnology Inc. (Santa Cruz, CA, USA). Polyclonal aac at-30) and ERaP3 (50-ggc att acc act tct cct ggg agt ct-30) antisera for mouse pituitary hormones (adrenocorticotropin was used to determine whether or not exon 3 had been K hormone (ACTH), GH, PRL, follicle-stimulating hormone deleted (ERa ). The presence of Zp3 Cre recombinase was (FSH), LH, and TSH) were purchased from the National determined using primers Cre-P1 (50-gga cat gtt cag gga tcg Hormone and Pituitary Program (Harbor-UCLA Medical cca ggc g-30) and Cre-P85 (50-gtg aaa cag cat tgc tgt cac tt-30). Center, Torrance, CA, USA). GnRH, 17b-estradiol (E2), CCK-8s, and lorglumide were purchased from Sigma. Primary pituitary cell culture Molecular reagents were purchased from Invitrogen. Cell culture reagents including Dulbecco’s Modified Eagle’s Anterior pituitary lobes were dissected from 10-week-old Medium (DMEM), gentamicin, BSA, HEPES, trypsin, female C57BL/6 mice pituitaries after carbon dioxide trypsin inhibitor, and DNase I were purchased from Sigma. inhalation. Pituitary cells were isolated as described previously Other reagents including ITS (insulin 10 mg/ml, transferin (Kim et al.2000) with minor modification. Briefly, anterior 5.5 mg/ml, sodium selenite 6.7 ng/ml), fungizone, and fetal pituitary lobes were minced into small pieces in serum-free bovine serum were purchased from Gibco-BRL. media, digested with trypsin for 20 min at room temperature, and dispersed in solution containing trypsin inhibitor by repeated sucking and pushing using an 18 G needle and syringe. Animals and treatments After washing, cells were counted and plated onto poly-L- Animal handling procedures were carried out in accordance lysine-coated culture dish that contained medium (20 mM with the University of Kentucky Animal Care and Use HEPES and 0.3% BSA in DMEM) supplemented with 10% Committee. All mice used in this study have a C57BL/6 fetal bovine serum or charcoal-treated fetal bovine serum. Cells genetic background. For ovariectomy (OVX) and estrogen/ were incubated in a humidified incubator at 37 8Cwith5% vehicle treatment, mice were ovariectomized at 45 days of age.