Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide regulating gonadotropin biosynthesis and release in the anterior pituitary via specific receptors. GnRH and its receptor are not only expressed in hypothalamus and pituitary. Several previous studies have reported an extra hypothalamic origin of GnRH, as well as an extra pituitary presence of its receptor in the reproductive tract of some of the mammalian species (Bauer-Dantoin and Jameson, 1995; Singh et al. 2011). The immuno localization and expression of GnRH and its receptor were shown in ovary of the mice and rat (Bauer-Dantoin and Jameson, 1995; Singh et al. 2011). GnRH expression has been shown in oviduct of the rat (Sengupta et al. 2007), human (Casañ et al. 1999) and porcine (Li et al. 1993). The expression of GnRH receptor mRNA and GnRH receptor protein have been shown in the bovine oviduct and uterus (Singh et al. 2008) and human (Raga et al. 1998). There are also reports regarding the presence of GnRH receptors in the male and female gametes (Dekel et al. 1988; Minaretzis et al. 1995; Morales et al. 1999) and embryos (Casan et al. 1999). In the ovary, GnRH is considered to act in an autocrine or a paracrine manner to regulate steroidogenesis by exerting a stimulatory as well as an inhibitory effect on the production of steroid hormones and apoptosis in ovarian follicles and corpora lutea (Dubois et al. 2002; Ramakrishnappa et al. 2005). The presence of GnRH in gametes indicated that it may have an effect on fertilization. Addition of GnRH to human semen increases the binding ability of sperms to the zona pellucida surrounding oocytes (Morales, 1998). In humans, GnRH and GnRH-receptor mRNA and protein expressions have been shown in endometrium throughout all phases of the menstrual cycle (Raga et al. 1998). The stimulatory effects of GnRH on cleavage cell divisions during early embryonic development and implantation have been reported in human (Tesarik et al. 2004). The presence of GnRH and the relative expression of its mRNA in the oviduct of pregnant rat (Sengupta et al. 2007) have led to the conclusion that it may have a possible role in post implantation embryonic development and the maintenance of pregnancy (Sengupta et al. 2007). GnRH and GnRH receptor mRNA are also expressed in placenta (Lin et al. 1995; Raga et al. 1998) and might affect the secretion of human chorionic gonadotropin (hCG). Addition of GnRH into the placental explant culture increased the secretion of hCG (Lin et al. 1995). The regulation mechanism of GnRH and its receptor in the peripheral reproductive organs is not well known. Estradiol-17β (E2) is major regulator of GnRH and GnRH receptor expression. Therefore, the aim of this study is to review the structure, function and expression of GnRH and its receptors in the peripheral reproductive organs and their regulation by E2.
Structure and function of GnRH-1
It was firstly proposed by Geoffrey Harris, John Everett, and Charles Sawyer that the hypothalamus controls reproductive function and a neurochemical signal from the hypothalamus is released into the anterior pituitary gland to stimulate gonadotropin secretion between 1930-1940 (Terasawa et al. 2010). Therefore, there must be a capillary portal vessel network connecting the hypothalamus to the pituitary. After a series of experimentations including the injection of Indian ink into the capillary network, Geoffrey Harris (Harris, 1948) demonstrated the presence of a portal vessel system in 1940 s. The hypothalamic factor controlling the pituitary secretion of gonadotropins was initially named as luteinizing hormone-releasing hormone (LHRH) because of its preferential positive effect on luteinizing hormone (LH) secretion rather than the secretion of follicle stimulating hormone (FSH) (McCann et al. 1960). However, injection of a specific LHRH antagonist suppressed both LH and FSH secretion. Therefore, it was named as gonadotropin releasing hormone (GnRH). Following studies focused on the extraction and the purification of GnRH, which was firstly purified from pig, ovine and bovine hypothalamus (Kochman and Domański, 1969; Schally et al. 1971). The molecular structure of GnRH was first explained by Andrew Schally and his team in 1971 (Schally et al. 1971). This GnRH is accepted as mammalian GnRH and designated as GnRH-I. The discovery of GnRH led to extensive research in this field and it is still an active field of research. This is because it is an important peptide for the regulation of the hypothalamic-pituitary-gonadal axis, and because recent advances in molecular biology have enabled the researchers to study its functions at multiple functional levels. The mammalian GnRH (GnRH-I) is consisted of ten amino acids as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 respectively. The length of the peptide and the amino acids both at the amino and at the carboxyl-terminal domains, which are important for receptor binding and activation, are highly conserved. In all vertebrate GnRH, glycine at the position 6 is strictly conserved. This amino acid is important for βII-type turn conformation of GnRH to bind its receptor. A three-dimensional structure of mammalian GnRH based on recent nuclear magnetic resonance (NMR) report showing the βII-type turn conformation around the glycine in position 6 (Figure 1).
Figure 1 NMR structure of mammalian GnRH showing the βII-type turn conformation around glycine in position 6 (obtained from the Figure 6 of Millar et al. 2008)
It is known that GnRH is synthesized from a larger prohormone by an enzymatic process and packaged into storage granules in the neurons in the preoptic area of the hypothalamus, then transported down to the axons located in the external zone of the median eminence (Kaiser et al. 1997; Millar et al. 2004; Cheng and Leung, 2005). It is released in a pulsatile manner, in synchronized pulses repeating every 30-120 minutes, from the nerve endings of about 1000 neurons into the hypophyseal-portal circulation system to the anterior pituitary, where it binds to its cognate-receptor on pituitary gonadotropin-secreting cells to control the secretion and synthesis of LH and FSH (Conn and Crowley, 1994; Stojilkovic and Catt, 1995; Sealfon et al. 1997). The amount of secreted gonadotropin depends on the amount of the GnRH reaching to the pituitary and the response of pituitary to GnRH. It is well known that the hypothalamus and the pituitary are not only the sites where GnRH and its receptor are expressed. Both GnRH and its receptor expression have been reported in the peripheral reproductive tissues and organs such as testis, ovary, oviducts, endometrium, placenta and in mammary glands of various vertebrate species (Siler-Khodr and Khodr, 1979; Khodr and Siler-Khodr, 1980; Oikawa et al. 1990; Li et al. 1993; Bahk et al. 1995; Raga et al. 1999; Harrison et al. 2004; Ramakrishnappa et al. 2005). As mentioned above, GnRH is synthesized from a larger prohormone by an enzymatic process. The gene encoding the preprohormone was first described in humans, which contains four exons and three introns (Figure 2).
Figure 2 The gene structure of human GnRH-I prohormone. It consists of four exons interrupted by three introns. Exon 1 encodes the 5'-UTR. Exon 2 encodes the signal peptide, GnRH decapeptide and the N-terminus of GAP. Exon 3 encodes the central portion of GAP and exon 4 encodes the C terminus of GAP along with the 3'-UTR (taken from Cheng and Leung, 2005)
The first exon of the gene is untranslated and consists of 61 bp in mRNA expressed in the hypothalamus. The second exon encodes the signal sequence, the GnRH decapeptide and GnRH-associated peptide (GAP) residues. The third exon encodes for the next GAP residues. The fourth exon encodes the remaining GAP residues and contains the translation termination codone and also the entire 3’-UTR (Adelman et al. 1986; Radovick et al. 1990). According to the pulse frequencies of GnRH, both LH and FSH are secreted by the same cells of pituitary known as gonadotrophs. Higher pulse frequency primes the secretion of LH, while lower frequencies prime the secretion of FSH (Dalkin et al. 1989; Haisenleder et al. 1993; Burger et al. 2002). External administration of GnRH results in LH and FSH secretion within 30 minutes. In peripheral tissues, such as in the ovary, GnRH affects steroid hormone production and in the placenta, it stimulates the secretion of chorionic gonadotropin (Hsueh and Schaeffer, 1985; King and Millar, 1995). A second form of GnRH was discovered in the chicken brain, and named as chicken-GnRH or GnRH-II. It is highly conserved peptide and also expressed in the central nervous system of many mammalian species including monkey (Lescheid et al. 1997) and human (White et al. 1998). Its amino acid sequence (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2) is 70% similar to that of GnRH-I differing in three amino acids (at positions 5, 7 and 8), but is encoded by a different gene (White et al. 1998) and is expressed by a distinct population of cells (Latimer et al. 2001). Initially, it was thought that GnRH-II promotes LH secretion in birds, but studies have shown that it can also promotes LH secretion in mammalians with a much lower potency than GnRH-I (Millar and King, 1983; Hasegawa et al. 1984). The expression and immune reactivity of GnRH-II is not only present in hypothalamus and pituitary gland, but also present in peripheral reproductive tissues of mammals including ovary, oviduct, uterus and placenta (Millar, 2003; Neill et al. 2001; Cheon et al. 2001; Siler-Khodr and Grayson, 2001; Choi et al. 2001; Chen et al. 2002). In addition to GnRH-I and GnRH-II, at least 45 structurally different forms of GnRH have been identified. Fifteen structural variants of the GnRH molecule have been found in vertebrates, and 15, in invertebrates (Millar et al. 2004; Roch et al. 2011), nine different GnRHs were identified in prochordates, which are vertebrate progenitors (Adams et al. 2003; Millar et al. 2004). A further six GnRH sequences were determined in other invertebrates. Gonadotropin-releasing hormone and its analogues have been used for the stimulation of gamete and hormone production, inhibition of ovulation and spermatogenesis, manipulation of puberty, synchronization of estrous in cattle, treatment of cystic ovary problem, treatment of uterine lesions and as well as used for the treatment of cancer.
Structure and function of GnRH receptor
Gonadotropin-releasing hormone (GnRH) acts via G-protein-coupled receptors (GPCRs), which are a family related to the rhodopsin and β-adrenergic receptors. GPCRs are known as the largest family of signaling proteins preferentially coupled to the Gq/11 protein localized in the cytoplasm and associated with the intracellular domains of the receptor (Stojilkovic et al. 1994). G-protein-coupled receptors are characterized by a hydrophilic extracellular N-terminal domain followed by hydrophobic seven transmembrane domains linked by a series of hydrophilic intracellular and extracellular loops and finally a hydrophilic intracellular carboxyl-terminal domain. The extracellular N-terminal domain contains ligand-binding and glycosylation sides as well as conserved cysteine residues forming disulfide bridges to stabilize the receptor structure (Figure 3). The seven transmembrane domains are known to be arranged in a tight bundle enclosing a hydrophilic pocket and surrounded by the hydrophobic membrane environment and believed to be involved in conformational change associated with signal propagation (receptor activation), while the intracellular domains are involved in interacting with G-proteins and other proteins for intracellular signal transduction (Sealfon et al. 1997; Flanagan et al. 1997; Schertler et al. 1993; Naor et al. 1998; Baldwin, 1993; Donnelly et al. 1989; Ballesteros and Weinstein, 1992). Binding of the mammalian GnRH to its receptor occurs via pGlu1, His2, Arg8 GlyNH210 with cognate sites D98(Asp98), K121(Lysine121), D302(Asp302) and N102 (Asn102) in the receptor. The Arginine8 in GnRH is essential for high affinity binding and selectivity of the receptor, while its mutation to Glycine leads to very poor binding efficiency (Ballesteros et al. 1998; Sealfon et al. 1997; Illing et al. 1999; Millar et al. 1989; Millar and King, 1983; Flanagan et al. 1994). Arginine side chain is involved in a triad interaction with aspargine87 in the second transmembrane domain (TMD2) and with aspartate318 in the seventh transmembrane domain (TMD7) to stabilize the active conformation of the receptor (Ballesteros et al. 1998). In non-mammalian vertebrate species (catfish, frog, chicken, and goldfish) and non-human primates, GnRH receptors have a carboxyl-terminal extension containing potential phosphorylation sites on multiple serine or threonine residues (Neill, 2002; Millar, 2003). When GnRH binds to its receptor, the receptor couple via heterodimeric G protein to phospholipase C (PLC) and adenylyl cyclase (AC) followed by phosphorylation of serine or threonine residues within carboxyl-terminal region. This phosphorylation is typically rapid (seconds to minutes) and mediated by specific G-protein receptor kinases (GRKs), by second messenger-regulated kinases (e.g. protein kinase C (PKC) or PKA), or by casein kinases (Tobin et al. 1997; Hanyaloglu et al. 2001) leading to β-arrestin binding, which hinders G-protein binding and thereby prevents effector activation (Zhang et al. 1997; Ferguson, 2001) and leading to the internalization of GnRH-Rs via clathrin-coated vesicles (Jennes et al. 1983; Jennes et al. 1986; Conn et al. 1987). The formation of these vesicles is typically controlled by a dynamin collar, which separates the vesicle from the plasma membrane by pinching off (or stretching) the neck of the vesicle (Schmid, 1998). After internalization, the receptors are either recycled to the cell surface or proteolytically degraded in lysosomes (Lefkowitz et al. 1990; Dohlman et al. 1991). Mammalian GnRH receptors do not desensitized rapidly, and do not undergo agonist-induced phosphorylation or cause β-arrestin translocation. They do show agonist-induced internalization, but this process is much slower and is not influenced by expression of β-arrestin (Heding et al. 1998; Heding et al. 2000; Vrecl et al. 1998; Willars et al. 1999). When GnRH binds to its receptor (GnRH-R), the receptor is coupled via Gq/11 to phospholipase C1 (PLC1). PLC1 cleaves phosphatidylinositol 4, 5-bisphosphate (PIP2), producing inositol trisphosphate (IP3), which mobilizes Ca2+ and thereby acutely regulates gonadotropin exocytosis. It also yields diacylglycerol (DAG), which activates PKC, feeding in to mitogen-activated protein kinase (MAPK) regulation and consequent regulation of gene expression. The structure of mammalian GnRH receptor, from different animals, shows about 85% homology. The length of the receptor protein varies between the mammalian and the non-mammalian species. Its length is 327-328 amino acids in cow, sheep and in human, while it is 370 amino acid-long in catfish due to the presence of 49 amino acids in the carboxyl-terminal domain, which is not present in mammalian receptors (Tensen et al. 1997). The expression both of the GnRH receptor mRNA and GnRH receptor protein are not only localized in pituitary gonadotrophs, but also localized in the peripheral reproductive tissues such as ovary, oviduct, uterus of mammals (Singh et al. 2008; Hsueh and Jones, 1981; Imai et al. 1994; Minaretzis et al. 1995; Borroni et al. 2000). GnRH and GnRH receptors are also found in many gonadal steroid-dependent cancers, including those of the breast, endometrium, prostate and ovary (Eidne et al. 1985;Miller et al. 1985;Emons et al. 1998;Limonta et al. 1999; Limonta et al. 2001;Schally, 1999; Schally and Nagy, 1999;Imai and Tamaya, 2000; Grundker et al. 2001). In humans, the GnRHR gene is located at 4 q 13.2 ± 3 and consists of three exons and two introns encoding a 328 amino acid protein (Figure 4). The proximal 5’-flanking region of the human GnRH receptor gene exhibits a great homology with that of the rodent and ovine sequences (Albarracin et al. 1994; Fan et al. 1995; Campion et al. 1996; Reinhart et al. 1997). But, there are certain specific differences in the structure of the gene between the human and rodent, such as the differences in transcription starting side and the differences in presence of multiple TATA and CAAT boxes.
Expression of GnRH and GnRH receptor in the peripheral reproductive organs
The expression of GnRH and GnRH receptor are not peculiar to hypothalamus and pituitary, they are expressed in the peripheral organs and tissues. The determination of peripheral expression of GnRH and GnRH receptor dates back to the late 1970s in rodent species. Specific radioligand binding sites were identified on rodent ovarian ganulosa and luteal cells (Clayton et al. 1979; Harwood et al. 1980; Reeves et al. 1980; Jones et al. 1980; Pieper et al. 1981). By using in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) techniques, the localization of GnRH mRNA and GnRH receptor mRNA in human ovarian granulosa and luteal cells has been shown (Minaretzis et al. 1995; Olofsson et al. 1995; Kang et al. 2000). Bu using RT-PCR and monoclonal GnRH receptor antibody through immunoblotting (Singh et al. 2008), the expressions of GnRH receptor mRNA and GnRH receptor protein have been shown in ovary, oviduct and in the uteri of bovine (Singh et al. 2008), porcine (Li et al. 1993) and human (Hsueh and Jones, 1981; Imai et al. 1994; Minaretzis et al. 1995; Ikeda et al. 1996; Dong et al. 1998; Raga et al. 1998; Borroni et al. 2000; Casañ et al. 2000). The presence of GnRH or a GnRH-like substance has also been reported in the placenta (Khodr and Siler-Khodr, 1978; Tan and Rousseau, 1982) and mammary glands (Amarant et al. 1982) of humans. Nucleotide sequence analyses in the rodent, bovine and human have shown that ovarian GnRH and GnRH receptor have sequence identical to those found in the hypothalamus and the pituitary (Kakar et al. 1992; Peng et al. 1994; Moumni et al. 1994; Olofsson et al. 1995; Whitelaw et al. 1995; Kottler et al. 1999). GnRH is regarded as an important paracrine and autocrine factor in the ovary. It has both inhibitory and stimulatory effects on ovarian function. GnRH exerts a stimulatory action on preovulatory follicles by inducing oocyte maturation (Hillensjo and LeMaire, 1980) and follicle rupture (Ekholm et al. 1981). On the other hand, GnRH has inhibitory effect on steroidogenesis involving the suppression of gonadotropin receptors (Hsueh and Jones, 1981) or the suppression of activity of the intermediary enzymes involved in steroidogenic pathway. It was also suggested that GnRH affects the process of fertilization and the cleavage rate of bovine oocytes in vitro (Funston and Seidel, 1995).
Figure 3 The mammalian GnRH receptor consists of an extracellular N-terminal domain followed by seven transmembrane domains linked by 3 intracellular and 3 extracellular loops. Mammalian GnRH receptor has no intracellular carboxyl-terminal domain. The extracellular N-terminal domain contains ligand binding and glycosylation sides as well as conserved cystein residues forming disulfide bridges to stabilize the receptor structure. GnRH binding sides are indicated in red and the sides are thought to be important in receptor structure or binding pocket configuration are indicated in green, including disulfide bond formation and glycosylation sites. Residues involved in receptor activation are shown in blue. Residues involved in coupling to G proteins are shown in orange. Putative protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites are indicated (obtained from Millar et al. 2004)
Figure 4 GnRHR gene consists of three exons and two introns. Exon 1 contains the 5’-UTR and encodes the first three transmembrane (TM) domains and a portion of the fourth TM domain. Exon 2 is 220 bp in length and encodes the remainder of the fourth TM domain, the fifth TM domain, and part of the third intracellular loop. Exon 3 encodes the rest of the open reading frame and contains the 3’-UTR (obtained from Cheng and Leung, 2005)
Effects of estradiol on peripheral GnRH and GnRH receptor expression
Estrogen is naturally occurring hormone within the body. Animal body naturally produces three main forms of estrogen, which are estradiol-17β (E2), estrone (E1) and estriol (E3). Estrone and estriol were first identified in the urine of pregnant women and this was followed by the identification of E2 in the follicular fluid of sow by Edward Adelbert Doisy between 1929 and 1936 (Simoni et al. 2002). Estradiol-17β is named for its importance in the estrous cycle affecting growth, development, maturation and functioning of reproductive tract, as well as the sexual differentiation and the behavior (Lien et al. 1985; Laugier et al. 1988; Balthazart et al. 2009). Two receptors, known as ERα (ESR1) and ERβ (ESR2), mediate effects of estrogen (Calatayud et al. 2010). Both are members of a large super family of proteins functioning as ligand-activated transcription factors (Katzenellenbogen and Katzenellenbogen, 1996). Their presence have been shown within the hypothalamus, pituitary, ovary, oviduct, uterus, cervix and vagina of mammalians including the human (Brodowskaet al. 2007), sheep (Juengel et al. 2006), cow (Sağsöz, 2011), goat (Cui et al. 2009), Porcine (Knapczyk-Stwora et al. 2011), rat (Okada et al. 2003) and mouse (Hułas-Stasiak and Gawron, 2007). It is clearly known that estrogen is a major regulator of GnRH neuronal function in the female brain and it has a bimodal effect on the hypothalamic-pituitary axis in females with both an inhibitory (Caraty et al. 1989; Sarkar and Fink, 1980; Levine and Ramirez, 1980; Chongthammakun and Terasawa, 1993; Evans et al. 1994; Evans et al. 1995) and stimulatory effect on GnRH and gonadotropin secretion. The stimulatory effect of estrogen on GnRH secretion is best illustrated at the end of the follicular phase where a gradual and sustained rise in circulating estrogen levels exerts a positive feedback effect on the hypothalamus triggering a preovulatory GnRH surge which, in turn, stimulates the preovulatory LH secretion (Moenter et al. 1990; Sarkar et al. 1976). Throughout the remainder of the cycle, estradiol exerts negative feedback actions on the central reproductive axis. GnRH is locally expressed in peripheral reproductive organs (Okrasa et al. 2003) and its expression is affected by estradiol. A study in ovariectomized (OVX) gilts showed that intramuscular injection of estradiol benzoate (EB) caused fluctuations in GnRH content according to the part of the reproductive tract. Estradiol benzoate treatment stimulated GnRH content in the ampulla of the oviduct and in the paracervical uterus, while inhibited GnRH content in the middle part of the uterus (Okrasa et al. 2003). The presence of GnRH mRNA have been shown in the human endometrium, while absence of mRNA for GnRH receptors have been shown by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis (Ikeda et al. 1997). Data related to the effect of estradiol on GnRH gene expression is limited. In vivo studies conducted in several mammalian species have indicated that estrogen reduces GnRH gene expression (Zoeller and Young, 1988; Petersen et al. 1995; Spratt and Herbison, 1997). Studies in postmenopausal women have shown that they have significant higher levels of GnRH mRNA when compared to premenopausal women (Rance and Uswandi, 1996). This suggests that lack of estrogen in postmenopausal women might have contributed to the high GnRH mRNA levels observed. Another study in adult female macaques indicated that ovariectomy significantly increased the number of GnRH-I expressing cells in the medial basal hypothalamus when compared to ovariectomized-estradiol treated animals (Figure 5, panel A). However, the number of GnRH-II expressing cells in ovariectomized animals was significantly lower than the number of the ovariectomized and estradiol treated group (Figure 5 panel B), Densmore and Urbanski (2004). The similar results were obtained from in vitro studies in human and rat. Treatment of human granulosa luteal cells with estradiol significantly decreased GnRH-I mRNA levels in a time-dependent manner, with maximum inhibition of 77% at 48 h (Khosravi and Leung, 2003). In rat hypothalamic explants, estrogen reduced GnRH mRNA expression (Wray et al. 1989). The studies given above indicate that estrogen negatively regulates GnRH gene expression; however, there is limited amount of information regarding the mechanisms of this negative regulation. Estradiol-17β (E2) is the major regulator of GnRH receptor gene expression and number during the preovulatory period. Treatment of ewes with estradiol caused significant increase in the concentrations of GnRH receptor mRNA and GnRH receptors on pituitary gonadotrophs (Turzillo et al. 1998).
Figure 5 The illustration of the effect of estradiol on GnRH-I and II gene expressions in the medial basal hypothalamus of ovariectomized (Ovx) adult rhesus macaques. Ovariectomy caused a significant increase in the number of cells expressing GnRH-I, while the number of cells expressing GnRH-II decreased. Estradiol treatment of ovariectomized animal (Ovx+E) caused decrease in the number of GnRH-I expressing cells, while the number of GnRH-II expressing cells increased (taken from the figure 2 of Densmore and Urbanski, 2004)
In a previous experimental study (Nathwani et al. 2000), human granulosa-luteal cells (hGLCs) were supplemented with different concentrations of estradiol (1-100 nM). It was reported that a short-term treatment (6 h) with E2 significantly increased GnRH receptor mRNA levels by 20%, whereas long-term treatment (48 h) resulted in a 60% decrease in GnRH receptor expression in hGLCs (Nathwani et al. 2000).
GnRH and GnRH receptor are also expressed in peripheral reproductive organs and estradiol-17β has negative effect on GnRH mRNA expression, but the exact mechanism has not been elucidated yet. The existing data only indicate that estradiol regulate GnRH receptor expression, but it does not clearly indicate that whether estradiol affects the process either positively or negatively.