Identification of a Membrane Estrogen Receptor in Zebrafish with Homology to Mammalian GPER and Its High Expression in Early Germ Cells of the Testis1

Abstract

To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [(3)H]-estradiol binding to plasma membranes of gper-transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper-transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.

Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis

“…7 mM) was shown to be rapid (within 5 min) and was transduced by a membrane-associated ER (Loomis & Thomas 2000). Recently, a membrane associated ER, homologous to mammalian GPER, has been cloned from zebrafish, and is expressed in testis (Liu et al 2009). The E 2 concentration used in the current study (10 nM) could have been sufficient to activate the zebrafish Gper.…”

“…However, the direct effects of oestrogen in the zebrafish testis were comparatively weak and possibly mediated via a nuclear Er, considering the prolonged time required for them to become evident, while the binding of E 2 to zebrafish Gper, although of high affinity (K d Z2 . 8 nM), was characterized by both rapid association and dissociation (Liu et al 2009).…”

Estrogen Signaling through the G Protein–Coupled Estrogen Receptor Regulates Granulocyte Activation in Fish

“…We observed that, in the tissues analyzed of gilthead seabream, the higher expression of GPER expression was observed in liver and gonad. In fish, GPER has been detected in Atlantic croaker and zebrafish gonads, and its activity has been related with the maturation of oocytes (51)(52)(53). Considerably, GPER is expressed in spleen and HK, the main hematopoietic organ in fish.…”

International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

“…Centrifugation methods have been employed for the preparation of subcellular fractions containing GPER that exhibit specific binding with filtration assays typically used to separate free from bound ligand in membrane fractions. This approach was first demonstrated in ER-negative cell lines using the human wild-type and recombinant receptor in SKBR3 and HEK293 cells, respectively , and later in human urothelial cell membranes (Teng et al, 2008) and with the zebrafish ortholog (Liu et al, 2009b). The lability of GPER requires short incubation times, low temperature (4°C), and the inclusion of protease inhibitors for reproducible ligand binding assays (Filardo and Thomas, 2012).…”

“…The lability of GPER requires short incubation times, low temperature (4°C), and the inclusion of protease inhibitors for reproducible ligand binding assays (Filardo and Thomas, 2012). Measured K d values for 17b-estradiol binding to GPER in membrane preparations from divergent species including zebrafish (Liu et al, 2009b), croaker (Pang et al, 2008), and recombinant human are very similar (2.3-3.3 nM) (Filardo and Thomas, 2012). The description of [ 3 H]E2 binding to GPER in membrane preparations as "limited capacity" Pang et al, 2008) likely reflects the low abundance or instability of the receptor in such preparations but has no functional implications.…”