Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility?

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Progress in Histochemistry and Cytochemistry xxx (2014) xxx–xxx www.elsevier.de/proghi

Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Marília I. Figueira 1 , Henrique J. Cardoso 1 , Sara Correia, Cláudio J. Maia, Sílvia Socorro ∗ CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal Received 26 July 2014; accepted 1 September 2014

Abstract The c-KIT, a tyrosine kinase receptor, and its ligand the stem cell factor (SCF) play an important role in the production of male and female gametes. The interaction of SCF with c-KIT is required for germ cell survival and growth, and abnormalities in the activity of the SCF/c-KIT system have been associated with human infertility. Recently, it was demonstrated that gonadotropic and sex steroid hormones, among others, regulate the expression of SCF and c-KIT in testicular and ovarian cells. Therefore, the hormonal (de)regulation of SCF/c-KIT system in the testis and ovary may be a cause underpinning infertility. In the present review, we will discuss the effects of hormones modulating the expression levels of SCF and c-KIT in the human gonads. In addition, the implications of hormonal regulation of SCF/c-KIT system for germ cell development and fertility will be highlighted. © 2014 Published by Elsevier GmbH.

Keywords: Androgens; c-KIT; estrogens; gonadotropins; infertility

∗ Corresponding author. CICS-UBI – Centro de Investigac ¸ ão em Ciências da Saúde, Faculdade de Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Tel.: +351 275329053. E-mail address: [email protected] (S. Socorro). 1 Both authors contributed equally.

http://dx.doi.org/10.1016/j.proghi.2014.09.001 0079-6336/© 2014 Published by Elsevier GmbH.

Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001

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Contents 1. 2.

3.

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5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brief overview of SCF/c-KIT distribution and function in the gonads . . . . . . . . . . . . . . . . . 2.1. Testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ovary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gonadotrophic actions regulating the expression of SCF and c-KIT . . . . . . . . . . . . . . . . . . . 3.1. Gonadotropin-Releasing Hormone (GnRH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Gonadotropins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of sex steroid hormones modulating the expression levels of SCF and c-KIT . . . . . . 4.1. Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other hormones playing a role in the regulation of SCF/c-KIT system . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The c-KIT is a type III receptor tyrosine kinase (Yarden et al., 1987) that specifically binds the stem cell factor (SCF), a membrane-bound cytokine also known as KIT ligand, steel factor or mast cell growth factor (Williams et al., 1990; Zsebo et al., 1990). Both c-KIT and SCF proteins display distinct isoforms, which differ, among other aspects, by its location at cell membrane or cytoplasm (Fox et al., 2000; Zhang et al., 2013). A cytoplasmic truncated form of c-KIT (tr-KIT), with only a part of the kinase domain and the carboxylterminal tail (Fig. 1A), is originated by a mechanism of alternative promoter usage (Rossi et al., 1992; Toyota et al., 1994). Moreover, the c-KIT protein can be proteolytically cleaved originating a soluble isoform (s-KIT, Fig. 1A) (Broudy et al., 1999; Turner et al., 1995). The SCF is present at cell membrane as a noncovalent homodimer (mSCF) (Lu et al., 1991; Matous et al., 1996; Zhang et al., 2000), and the proteolytic cleavage of an alternatively spliced variant of SCF originates its soluble isoform (sSCF, Fig. 1B) (Flanagan et al., 1991; Majumdar et al., 1994; Pandiella et al., 1992). The interaction of SCF with c-KIT leads to dimerization of receptor, activation of its tyrosine kinase activity and initiation of downstream signal transduction pathways (BlumeJensen et al., 1991). The SCF/c-KIT system has been shown to play an important function in melanogenesis, hematopoiesis and gametogenesis (Nishikawa et al., 1991; Ratajczak et al., 1992; Sato et al., 2012) through the regulation of several biological processes, such as cell proliferation, differentiation, migration and apoptosis (Farini et al., 2007; Ronnstrand, 2004). Moreover, deregulated actions (or abnormal expression levels) of SCF and c-KIT have been associated with reproductive disorders and infertility (Blume-Jensen et al., 2000; Ciraolo et al., 2010; Sandlow et al., 1996). Human fertility depends on several psychological, physical and biochemical factors, including the intricate and complex hormonal regulation that governs the production of germ cells. Spermatogenesis and oogenesis, the cellular processes that originate, respectively, male and female gametes, are coordinated by a set of hormones and paracrine factors in Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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Fig. 1. Membrane, cytoplasmic and soluble isoforms of c-KIT (A) and SCF (B). (A) The c-KIT protein contains three functional domains: i) the extracellular ligand-binding domain containing the five typical immunoglobin-like domains (blue); ii) the transmembrane domain (brown); iii) the cytoplasmic domain harboring the proximal and distal kinase domains, separated by an interkinase region (green), and a carboxyl terminal tail (black). c-KIT protein displays alternatively spliced forms characterized by the presence or absence of the tetrapeptide Gly-Asn-Asn-Lys (GNNK) in the extracellular juxtamembrane domain (c-KIT 1 and c-KIT 2, respectively). The c-KIT can be proteolytically cleaved originating a soluble isoform (s-KIT) released to the extracellular space. A mechanism of alternative promoter usage generates a cytoplasmic truncated isoform of c-KIT (tr-KIT), which misses the extracellular domain, the transmembrane region and part of the kinase domain. (B) The SCF, the ligand for c-KIT, exists as a membrane-bound isoform (mSCF) or it may suffer proteolytic cleavage originating a soluble SCF isoform (sSCF).

response to the activity of the hypothalamic-pituitary-gonadal axis (Schlatt and Ehmcke, 2014). Interestingly, recent evidence has been indicating that both pituitary and gonadal hormones, among others, regulate the tissue expression levels of SCF and c-KIT (Correia et al., 2014; Diallo et al., 2006; Majumdar et al., 2012). The present review aims to address the hormonal influences on the regulation of SCF/c-KIT system discussing the physiological impact for male and female fertility.

2. Brief overview of SCF/c-KIT distribution and function in the gonads 2.1. Testis The mammalian testis is divided into two compartments, the seminiferous tubules, where spermatogenesis takes place, and the interstitial space (Russell, 1990). The expression of Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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SCF in the testis is essentially restricted to Sertoli cells (Sandlow et al., 1996), the somatic cells within seminiferous tubules, which promote and sustain the development of male germ cells (Mruk and Cheng, 2004). On the other hand, developing germ cells specifically express the c-KIT receptor (Unni et al., 2009), which has been indicated as a robust marker of the germ cell population (Gaskell et al., 2004; Sa et al., 2013). Therefore, the germ cells expressing c-KIT are influenced by the somatic Sertoli cells, which present SCF at their plasma membrane (Correia et al., 2014; Unni et al., 2009). This germ cell: Sertoli cell communication mediated by the SCF/c-KIT system has been indicated as a powerful mechanism determining the survival of germ cells (Bokemeyer et al., 1996; Yan et al., 2000a; Yan et al., 2000b). The activation of c-KIT by SCF is a crucial event protecting germ cells from apoptosis (Yan et al., 2000b), and is also needed for the onset and progression of spermatogenesis (Guerif et al., 2002; Sato et al., 2012). The c-KIT receptor is also detected in Leydig cells (Muciaccia et al., 2010; Unni et al., 2009), another somatic cell type of the testis, which are located in the interstitium between seminiferous tubules and are responsible for the secretion of testosterone (Haider, 2004). In addition, it has been suggested that the SCF/c-KIT stimulates the steroidogenic activity of Leydig cells increasing the production of testosterone (Rothschild et al., 2003). 2.2. Ovary The ovarian follicles are the functional units of mammalian ovary, which are formed by the oocyte and surrounding cells, namely, the granulosa and theca cells. Normal folliculogenesis encompasses the development of follicles through primordial, primary, secondary, antral and preovulatory stages (Makrigiannakis et al., 2008). In the fully-developed ovary, c-KIT is mainly expressed in oocytes whereas granulosa cells synthesize the SCF, which is widely recognized as a stimulator of oocyte growth (Parrott and Skinner, 1999). Indeed, the granulosa cells secrete all the nutritional factors required for oocyte growth (Matsuda et al., 2012). The ligand-receptor interaction, establishing a communication between oocytes and granulosa cells, is also involved in the development of primordial follicles, growth of primary follicles and emergence of pre-ovulatory follicles (Manova et al., 1993; Yoshida et al., 1997).

3. Gonadotrophic actions regulating the expression of SCF and c-KIT 3.1. Gonadotropin-Releasing Hormone (GnRH) The GnRH produced by the hypothalamus is responsible for the release of gonadotropins from the anterior pituitary and plays an important function in the onset of puberty, both in boys and girls. In adult men and women GnRH actions are the master controller of the pituitary-gonadal axis and reproductive function. For this reason, GnRH antagonists or agonists have been used to treat failures of reproductive development and function in humans as well as in animal models (Blanchard et al., 1998; Carranza et al., 2014; Dong Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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et al., 2011). Some of the beneficial effects of GnRH administration are coupled to the regulation of SCF levels in testicular and ovarian cells. The exposure of rats to the hexane metabolite 2,5-hexanedione (2,5-HD) has shown to induce irreversible testicular atrophy (Boekelheide, 1988; Chapin et al., 1983). The testicular injury induced by 2,5-HD is accompanied by the reduced expression of SCF and an altered localization of protein, shifting from a predominant distribution at plasma membrane to a soluble form (Allard et al., 1996). Treatment with the GnRH agonist leuprolide used as a therapy for 2,5-HD-dependent testicular atrophy, significantly increased the levels of mSCF (Blanchard et al., 1998). In Sertoli cells isolated from rat testis, treatment with GnRH agonists did not change the percentage of the mSCF (Plotton et al., 2005). However, the mRNA levels of SCF displayed a biphasic pattern increasing between days 0 and 21, and decreasing afterwards till day 28 (Plotton et al., 2005). Nevertheless, the presence of GnRH receptor has been described in Leydig cells and germ cells but not in Sertoli cells (Anjum et al., 2012; Bull et al., 2000; Reinhart et al., 1992). This suggests that factors secreted by Leydig cells or germ cells in response to GnRH may act on Sertoli cells regulating the expression of SCF. In the ovary, SCF is an important factor playing a role in follicle growth and activation of primordial follicles (Parrott and Skinner, 1999). Recently, the effect of GnRH agonist (triptorelin) and GnRH antagonist (cetrorelix) on the SCF expression in human granulosa cells was described. Treatment with 10−7 M of cetrorelix significantly increased SCF mRNA and protein expression, whereas treatment with triptorelin did not change the SCF levels (Dong et al., 2011). Thus, authors suggested that GnRH agonists and antagonists may have distinct effects on the expression of ovarian autocrine/paracrine factors such as SCF, differentially affecting the ovarian reserve (Dong et al., 2011; Torrealday et al., 2013). In sum, GnRH actions alter the expression of SCF, as well as its location at cell compartments, which seems to be related with the extent of both male and female germ cell populations. 3.2. Gonadotropins At puberty and throughout the reproductive years, the GnRH stimulates the anterior pituitary to secrete the gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH is widely recognized as the crucial hormone for initiation of spermatogenesis and follicular development (Richards et al., 2002; Zirkin, 1998). In the testis, FSH actions are exerted through FSH receptors present in Sertoli cells, and involve the regulation of SCF expression (Rossi et al., 1993; Yan et al., 1999). Experimental assays using the transcription inhibitor actinomycin and the inhibitor of translation cycloheximide indicated that the FSH regulation of SCF expression occurs at transcriptional level independently of de novo protein synthesis (Yan et al., 1999). It has been shown that the effect of FSH stimulating the expression of SCF depends on the maturation stage of Sertoli cells, and that the increased expression of SCF contributes to differentiation and maturation of spermatogonia (Feng et al., 2000). Prepubertal Sertoli cells of rodents respond positively to the FSH treatment by enhancing the mRNA expression of SCF, an effect absent in neonatal Sertoli cells (Bhattacharya et al., 2012; Rossi et al., 1993). In monkeys, it was verified that the FSH treatment inducing the expression of SCF in Sertoli cells is 5- to 10-fold Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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higher in pubertal testes comparatively with infantile testes (Majumdar et al., 2012). In vivo treatment with methoxy-acetic acid, which augments the levels of FSH, also increased the mRNA expression of SCF (Plotton et al., 2005). FSH treatment of chicken germ cells co-cultured with Sertoli cells induced proliferation of germ cells, which was concomitant with the upregulated expression of c-KIT (Mi et al., 2004). Although this study did not analyze the expression levels of SCF, the proliferative response of germ cells should be a consequence of the stimulatory effect on Sertoli cells with the increased production of growth factors including the SCF. In the ovary, FSH regulates the release of several paracrine factors and the oocyte: granulosa cells interaction during follicular development. The role of FSH in primordial folliculogenesis seems to occur via SCF because the number of FSH-stimulated primordial follicles decreases in presence of an SCF antibody (Wang and Roy, 2004). In fact, it was demonstrated that FSH enhances the expression of SCF in mice and bovine granulosa cells (Parrott and Skinner, 1998; Thomas et al., 2005). However, a dual effect was seen in the case of mice granulosa cells. Low doses of FSH increased the expression of mSCF decreasing the ratio soluble/membrane form. On the other hand, high levels of FSH increased the ratio sSCF/mSCF (Thomas et al., 2005). Although both doses of FSH increased the diameter of oocyte-granulosa cell complexes, only low doses promoted oocyte growth, and this growth was inhibited when c-KIT was blocked. Interestingly, when sSCF was exogenously administrated, the FSH-stimulated oocyte growth was suppressed, suggesting that the growth effects are dependent on the ratio of sSCF/mSCF (Thomas et al., 2005). It was also demonstrated that the FSH regulation of oocyte growth involves an interaction between granulosa and theca cells, which is mediated by the action of SCF. It was shown that upon stimulation with FSH, bovine granulosa cells actively secrete SCF (Parrott and Skinner, 1998). The theca cells express the keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) genes and secrete proteins that can regulate the granulosa cell growth. In fact, SCF stimulates the expression of KGF and HGF in theca cells, and these, in turn, stimulate the mRNA expression of SCF in granulosa cells. So, FSH can stimulate the granulosa cells to release paracrine factors, such as SCF (Fig. 2), and it exerts a positive feedback on oocyte development and in the regulation of theca cells (Parrott and Skinner, 1998). Corroborating all these data is the information on knockout mice for FSH receptor, which displayed disrupted communication between oocyte and granulosa cells, altered follicular development and under-expression of SCF and c-KIT (Yang et al., 2003). The gonadotropin LH stimulates the Leydig cells to produce testosterone, which together with FSH stimulates the spermatogenic process (O’Shaughnessy, 2014). However, the relationship between LH and the expression or activity of the SCF/c-KIT system in mammalian testes is unknown. Indeed, the information is scarce regarding the role of both gonadotropins (LH and FSH) on the regulation of c-KIT levels in the male gonad. Interestingly, an abnormal pattern of c-KIT expression was shown in the fetal gonads of intersex subjects. Germ cells isolated from gonads with abnormal development and sex differentiation displayed stronger expression of c-KIT comparatively with normal controls at a similar developmental age (Rajpert-De Meyts et al., 1996). As these authors suggest this may indicate that the expression of c-KIT is regulated by factors that control the differentiation of gonads. The actions of LH in female reproduction are essentiality related with the induction of ovulation and maintenance of the activity of corpus luteum (Jones and Lopez, 2014), Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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Fig. 2. Hormonal regulation of SCF/c-KIT system in the testis (A) and ovary (B). (A) The GnRH seems to increase the production of SCF likely through paracrine factors secreted by Leydig cells, which stimulate the Sertoli cells. The sex steroids estrogens increase the activity of c-KIT while downregulating its expression levels. The GHRH, and the synergistic action of androgens with FSH enhance the levels of SCF in Sertoli cells. The action of SsT in the regulation of SCF/c-KIT system can occur by two mechanisms: i) inhibition of FSH activity or ii) inhibition of c-KIT activity in germ cells. (B) GnRH antagonists are associated with an increased expression of SCF through a direct action at granulosa cells, which express the GnRHr. The effects of LH increasing the levels of SCF in granulosa cells, also occur directly via the LHr present in this cell type. Alternatively, LH may act indirectly via theca cells, which secrete and release paracrine factors that stimulate the granulosa cells. FSH and androgens actions mediated by their cognate receptors present in the granulosa cells, lead to enhanced production of SCF. In turn, theca cells may increase the production of androgens in response to the activity of c-KIT. Increased levels of estrogens diminish the production of SCF by granulosa cells. AR, androgen receptor; ER, estrogen receptor; FSH, follicle-stimulating hormone; FSHr, follicle-stimulating hormone receptor; GHRH, growth-hormone-releasing hormone; GHRHr, growth-hormone-releasing hormone receptor; GnRH, gonadotropin-releasing hormone; GnRHr, gonadotropin-releasing hormone receptor; LH, luteinizing hormone; LHr, luteinizing hormone receptor; SsT, somatostatin; SsTr, somatostatin receptor; (-) GnRH, GnRH antagonists; ?, undetermined.

and there are studies describing the regulation of SCF and c-KIT by this gonadotropin. Treatment with LH agonists, such as pregnant mare serum gonadotropin (PMSG) or human chorionic gonadotropin (hCG), increased the expression of SCF in normal ovarian surface epithelium (Parrott et al., 2001) and granulosa cells (Ismail et al., 1996; Motro and Bernstein, 1993; Parrott and Skinner, 1998). The LH actions can be mediated directly by the LH receptor present in the granulosa cells (Robert et al., 2003), or indirectly through thecal cells, which express genes that regulate the activity of granulosa cells (Parrott and Skinner, 1998). Nevertheless, the increased expression of SCF in response to LH is linked to ovarian epithelial growth and follicular development (Ismail et al., 1996; Motro and Bernstein, 1993; Parrott et al., 2001; Parrott and Skinner, 1998). Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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Also, there are reports indicating the effect of LH on the regulation of c-KIT levels in ovarian cells. However, these studies have produced contradictory results. Although some studies indicated that LH decreases the expression of c-KIT in theca and interstitial cells (Motro and Bernstein, 1993), others have shown that LH is not able to modulate the c-KIT levels (Ismail et al., 1997). Further studies are needed to deeply understand the role of gonadotropic hormones modulating the expression of SCF/c-KIT in the ovary and testis, which may be an important clue to develop novel strategies for treatment of human infertility.

4. Role of sex steroid hormones modulating the expression levels of SCF and c-KIT 4.1. Estrogens Estrogens are the major players in female fertility due to its known actions regulating the ovarian and endometrial cycles. Estrogenic actions are mediated by the classic intracellular receptor proteins (estrogen receptors alpha (ER␣) and beta (ER␤)), which act as transcription factors regulating the expression of target genes (Gibson and Saunders, 2012). Alternatively, estrogens may elicit signalling events by interaction with the G-proteincoupled receptor-30 (GPR30/GPER) (Prossnitz et al., 2008). In mammals, oocytes and spermatozoa are originated from the primordial germ cells that migrate to the genital ridges and proliferate actively before birth (McLaren, 2003). It has been shown that estrogens stimulate the proliferation of primordial germ cells (La Sala et al., 2010; Moe-Behrens et al., 2003). This effect was suggested to occur in response to growth factors secreted by the gonadal somatic cells because primordial germ cells do not express ERs (Moe-Behrens et al. (2003). However, La Sala et al. (2010) showed that primordial germ cells express the ER␣, and demonstrated that treatment with 10−8 M 17␤-estradiol (E2 ) increased the proliferation/survival of primordial germ cells, which was underpinned by the enhanced phosphorylation of c-KIT. The SCF/c-KIT signalling pathway has been widely implicated in the control of reproductive functions, and its action is crucial to drive migration and proliferation of primordial germ cells (Blume-Jensen et al., 2000; De Miguel et al., 2002; Farini et al., 2007). Thus, not surprisingly, it was found that the estrogenic effects promoting the proliferation of primordial germ cells were somehow related with the expression and/or activity of SCF and c-KIT. Primordial germ cells express the c-KIT receptor whereas the gonadal somatic cells synthesize both sSCF and mSCF (Besmer et al., 1993). E2 treatment promoting proliferation of primordial germ cells was accompanied by a 4-fold increase in the expression of sSCF by gonadal somatic cells, which seems to occur by interaction of ER␣ with an AP-1 response element in the Steel gene encoding the SCF protein (Moe-Behrens et al., 2003). Moreover, the use of anti-SCF antibody abolished the E2 -induced proliferation, which supports that proliferative effects of estrogens are due to the activity of SCF/c-KIT system (Moe-Behrens et al., 2003). Indeed, in vitro stimulation of primordial germ cells with E2 lead to the phosphorylation of c-KIT (Fig. 2) and activation of downstream molecular targets (La Sala et al., 2010). Since ERs do not have intrinsic kinase activity, the E2 -induced Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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phosphorylation occurs indirectly after receptor association with other proteins. The phosphorylation of c-KIT in primordial germ cells in response to E2 is dependent on the activity of SRC tyrosine kinases likely through the action of an adaptor protein that mediates the interaction of ERs with SRC proteins (La Sala et al., 2010; Wong et al., 2002). The activation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target the serine/threonine kinase Akt are well-known effectors of c-KIT signalling (Blume-Jensen and Hunter, 2001). Moe-Behrens et al. (2003) demonstrated that the use of a potent inhibitor of c-KIT-dependent Akt activation (SU5416, 20 ␮M), and a specific inhibitor of PI3-K (LY294002, 10 ␮M), reduced the estrogen-dependent increase of primordial germ cells growth. This allowed authors to suggest that estrogenic actions on primordial germ cells require the c-KIT signalling with subsequent activation of PI3-K and Akt (Moe-Behrens et al., 2003). Considering that the growth of ovarian follicles is dependent of SCF/c-KIT signalling in the earlier stages of folliculogenesis, and the known influence of estrogens on this process, some studies have explored the effect of E2 regulating the expression of SCF and c-KIT in the ovary. The first evidence of a relationship between the expression levels of SCF/c-KIT and E2 appeared with the report of Tanikawa et al. (1998), which positively correlated the expression of c-KIT in human oocyte with the volume of fluid and the concentration of E2 in follicular fluid (Tanikawa et al., 1998). In the ovary of newborn mice, both in vitro and in vivo approaches demonstrated that E2 is able to regulate SCF expression (Fig. 2). The mRNA expression of SCF decreased in mouse ovaries cultured in vitro with 10-4 M of E2 for 2 and 4 days (Huansheng et al., 2011). Also, in the ovary of newborn mice injected with E2 (5 mg/kg/day) a decreased expression of SCF mRNA was observed at 2 and 4 days after administration (Huansheng et al., 2011). However, treatment with a 10-8 M dose of E2 for 4 days seems to induce an increase in the mRNA expression of SCF in mice ovary cultured in vitro (Huansheng et al., 2011), which indicates that estrogenic effects on the activity of SCF/c-KIT system may vary with the hormonal dose. Moreover, these experimental findings raise the concern about the consequence of environmental exposure to estrogens or estrogen-mimicking substances on the expression of SCF and c-KIT in the ovary. Premature ovarian failure is a condition characterized by the loss of normal ovary function before the age of 40, which leads to premature menopause and infertility. It may result from chromosomal abnormalities, chemo- or radiotherapy, or being associated with rare disorders (Cox and Liu, 2014). However, spontaneous ovarian failure in healthy women with normal karyotypes (46,XX) accomplishes for 1% of infertility cases in women < 40 years of age (Nelson, 2009). In the last years, a growing body of evidence has been indicating that endocrine-disrupting chemicals with estrogenic activity may contribute to the onset of this disorder (Craig et al., 2011; Gore et al., 2011). On the other hand, it is known that the SCF/c-KIT system plays a role in the primordial follicle activation and maintenance of oocyte dormancy (Hutt et al., 2006). Moreover, as exposed above, the expression levels of SCF and c-KIT in the ovary seem to be modulated by estrogens. Therefore, it is plausible to assume that estrogen disruptors mimicking the action of endogenous hormones can deregulate the expression of SCF/c-KIT in ovarian cells, and promote the early activation of follicle pool, which may cause the premature ovarian failure and infertility. Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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Although estrogens are classically view as female hormones, the last decades have witnessed the emergence of a set of experimental and clinical evidences indicating the importance of estrogens for production of viable spermatozoa, and thus for male fertility (Carreau and Hess, 2010; Hess, 2003). By contrast, a substantial amount of data also has ascribed the deleterious effects of estrogens and estrogen-like substances for male reproductive function. Abnormal exposure to estrogens has been shown to affect male sexual differentiation (Sultan et al., 2001; Toppari, 2002) and the spermatogenic output (Sweeney, 2002; Sweeney et al., 2007). In an attempt to investigate the regulatory factors controlling the expression of SCF during spermatogenesis, Yan and co-authors firstly investigated the effect of estrogens on the testicular expression of SCF/c-KIT system. Using a 10-8 M dose of E2 authors did not observe changes on the expression of SCF in rat seminiferous tubules cultured in vitro (Yan et al., 1999). Very recently, using a similar experimental approach other study demonstrated that a 10-7 M dose of E2 induced a decrease in c-KIT expression (Fig. 2) while increasing the expression of SCF (Correia et al., 2014). Moreover, the altered expression of the SCF/c-KIT system relied on increased apoptosis and decreased proliferation of germ cells (Correia et al., 2014). The importance of estrogens controlling testicular physiology via the regulation of c-KIT expression was further confirmed by another recent study using rats exposed to phytoestrogens. Twenty-one-day old male rats fed daily for 3 months with a standard diet supplemented with soya isoflavones (20 mg/kg/day) displayed decreased levels of testosterone and a diminished expression of c-KIT in spermatogonia, spermatocytes and spermatids, with important alterations in the morphology of seminiferous tubules (Misiakiewicz et al., 2013). Noteworthy, the deregulated expression of SCF and c-KIT has been reported in the testis of subfertile and infertile men (Bialas et al., 2010; Feng et al., 1999; Medrano et al., 2010; Plotton et al., 2006; Sandlow et al., 1996). A diminished expression of c-KIT was linked to increased apoptosis in the testes (Feng et al., 1999). Moreover, the decreased expression of c-KIT in the testes of azoospermic men was correlated with the severity of the disease, decreasing from cases of maturation arrest to cases of Sertoli cell only syndrome, which are characterized by the absence of germ cells (Bialas et al., 2010; Medrano et al., 2010). Although c-KIT is also present in Leydig cells, the decreased levels of c-KIT in testes with disrupted spermatogenesis seem to follow the reduction in the population of germ cells. Regarding SCF, an opposite pattern was observed. The expression of SCF augmented accordingly with the degree of spermatogenesis impairment (Feng et al., 1999; Sandlow et al., 1996), which may be a protective mechanism of Sertoli cells to counteract the loss of germ cells. As described above, estrogens downregulate the expression of c-KIT in seminiferous tubules concomitantly with diminished proliferation and enhanced apoptosis of germ cell (Correia et al., 2014). It is also known that hyperestrogenism is a hormonal deregulation associated with some cases of male idiopathic infertility (Lardone et al., 2010; Levalle et al., 1994; Marie et al., 2001). These findings support the idea that the elevated concentrations of estrogens are the cause underlying male infertility due to their effects on the reduction of c-KIT expression. To fully address this question, it will be of uttermost importance to study the expression of the SCF/c-KIT system in the testis of infertile men displaying a hormonal profile of hyperestrogenism. Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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4.2. Androgens Androgens are the most abundant sex steroids in men, which are responsible for the development of male organs and secondary sexual characteristics. The most well-known androgens are testosterone and its 5␣-reduced metabolite, the potent androgen 5␣dihydrotestosterone (DHT). Other common androgens include the dehydroepiandrosterone (DHEA), its metabolite the dehydroepiandrosterone sulfate (DHEAS) and androstenedione. Moreover, testosterone is the obligatory precursor in the biosynthesis of estrogens through its aromatization to E2 by the activity of aromatase enzyme. In the testis, synergistic actions of testosterone and FSH are absolutely required for development and survival of germ cells (Chang et al., 2004; O’Shaughnessy et al., 2012). However, the unleashing effects of testosterone and FSH in spermatogenesis are integrated via Sertoli cells since germ cells are devoid of androgen receptors (Walker and Cheng, 2005). Moreover, the SCF protein produced by Sertoli cells directly regulates the survival and proliferation of the germ cells, which express the SCF receptor, c-KIT (Rossi et al., 2000). Therefore, it became a relevant scientific question to explore whether testosterone regulates the expression of SCF and c-KIT in testicular cells. It was reported that in vitro culture of rat seminiferous tubules with testosterone (10-6 M) did not change the mRNA expression of SCF (Yan et al., 1999). Also, no significant changes were found on the expression of SCF in rat Sertoli cells at different developmental ages after treatment with testosterone (10-7 M) for 24 h (Bhattacharya et al., 2012). An identical response was described in Sertoli cells of rhesus monkey (Macaca mulatta) using the same hormonal doses and time-frame (Majumdar et al., 2012). However, the combined treatment of monkey Sertoli cells with the same concentration of testosterone plus FSH (5 ng/ml) increased the mRNA levels of SCF (Fig. 2) in pubertal Sertoli cells comparatively with infant Sertoli cells (Majumdar et al., 2012). Concerning the c-KIT there are evidences relating its expression with the phase of reproductive cycle and the serum levels of androgens. In the seasonal breeder green frog (Rana esculenta), the expression of c-KIT throughout reproductive cycle revealed a seasonal pattern linked with the plasma and testicular levels of testosterone. The maximum expression of c-KIT arose during the reproductive period when the testis exhibited the maximum concentration of testosterone (Raucci and Di Fiore, 2007). Similar results were seen in the Italian wall lizard (Podarcis sicula) (Raucci and Di Fiore, 2009) and also in the mammal roe deer (Capreolus capreolus) (Roelants et al., 2002). Since the c-KIT is highly expressed in germ cells it is expected that an increase in the spermatogenic output in response to enhanced production of testosterone may result in the enhanced expression of receptor as observed in the testis of mammalian and non-mammalian vertebrates. However, Leydig cells also express the c-KIT, and the SCF/c-KIT system seems to play a role regulating the proliferation and testosterone biosynthesis in these cells (Yan et al., 2000a). In this way, further studies are needed to determine whether androgens regulate the expression of cKIT in Leydig cells, which could represent an auto-regulatory mechanism controlling the testosterone production. However, androgens also seem to play an important role in female physiology, particularly in the ovarian function (Walters et al., 2008). A significant correlation was found between the expression of c-KIT and the concentrations of testosterone and Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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androstenedione in the follicular fluid (Tanikawa et al., 1998). In pregnant pigs, treatment with the anti-androgen flutamide (50 mg/kg body weight during seven days) decreased the expression of both c-KIT and SCF in the embryonic ovary (Knapczyk-Stwora et al., 2013). Moreover, after anti-androgen treatment, the fetal ovaries displayed a higher quantity of primordial follicles and lower quantity of primary follicles comparatively with the control group, which indicated an impairment of follicular progression (Knapczyk-Stwora et al., 2013). This suggests that the androgenic actions regulating the bioavailability of the SCF/c-KIT system may be involved in the transition of primordial to primary follicles. Accordingly, 3-week-old female mice injected with DHT showed a great induction of SCF expression in the ovary 4 h after treatment, an effect that was attenuated in the presence of flutamide (Shiina et al., 2006). The stimulatory effect of DHT on the expression of SCF was further confirmed in human granulosa-like tumor cells in culture (Shiina et al., 2006). On the other hand, testosterone treatment in vivo was ineffective regulating the expression of SCF in sheep follicles of any of the fetal or postnatal ages (Veiga-Lopez et al., 2012). Also in laying hens, treatment of 6–8 mm follicles with testosterone (0.1, 1, 10, 50 ng/ml) did not change the expression of SCF in granulosa cells (Kundu et al., 2012). However, in mural granulosa cells of antral follicles and in cumulus granulosa cells of mouse ovary, testosterone (1 ng/ml) significantly increased the expression of splice variants of SCF. Polycystic ovary syndrome (PCOS) is a common complex endocrine disorder characterized by the overproduction of androgens, which causes irregular or absent ovulations (Nisenblat and Norman, 2009). Despite intense research efforts, the etiology and the molecular mechanisms underlying the onset of PCOS remain unknown. As stated previously, androgens are able to modulate the expression levels of SCF and c-KIT in granulosa cells and oocytes, respectively. However, c-KIT is also expressed in the theca cells (Merkwitz et al., 2011), which are the androgen-producing cells in the ovary (Magoffin, 2005). The abnormal proliferation and activity of theca cells has been suggested as the primary source for hyperandrogenism (Magoffin, 2005), and Parrot and co-authors reported that the SCF/c-KIT system is capable of regulating the growth and differentiation of theca cells (Parrott and Skinner, 1998). Moreover, it was shown that c-KIT may control the steroidogenesis in ovarian and testicular cells (Miyoshi et al., 2012; Rothschild et al., 2003) increasing the biosynthesis of testosterone (Rothschild et al., 2003). Therefore, an androgenic effect stimulating the expression of c-KIT in theca cells, and in turn, the augmented production of androgens by theca cells in response to c-KIT (Fig. 2) may constitute a positive feedback loop mechanism driven the appearance of PCOS.

5. Other hormones playing a role in the regulation of SCF/c-KIT system Some studies have been indicating that the expression of SCF and c-KIT can be regulated by hormonal factors other than gonadotropins or sex steroids (Fig. 2). It is known that somatostatin regulates the levels of several hormones and is able to inhibit the activity of FSH in Sertoli cells (Krantic and Benahmed, 2000). Goddard and co-authors Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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demonstrated that somatostatin decreases the expression of SCF in pig immature Sertoli cells in culture (Goddard et al., 2001). Moreover, somatostatin abolishes the FSH-induced up-regulation of SCF expression in Sertoli cells, and the proliferation of spermatogonial cells in response to SCF (Goddard et al., 2001). The GHRH is the main hypophysiotropic hormone responsible for the synthesis of growth hormone and its release from the somatotrophs in pituitary (Mayo et al., 1995). The GHRH-related peptide (GHRH-RP), the carboxyl-terminal moiety of GHRH, stimulates the expression of SCF in rat Sertoli cells in culture (Breyer et al., 1996). Also, a transgenic mouse overexpressing the GHRH-RP presents overexpression of SCF (Fang et al., 2000). The GHRH-RP may undergo proteolytic modifications, resulting in the production of the p75-92NH2, a GHRH-RP-like peptide. This peptide seems to be active in Sertoli cells increasing the levels of cAMP similarly to what is observed after treatment with GHRH or GHRH-RP (Nillni et al., 1999). In fact, stimulation of Sertoli cells with the GHRH-RP increased the expression of SCF during 16 h; 24 h after treatment the SCF levels returned to values close to the control. However, this effect was slower comparatively with that of GHRH, which augmented the expression of SCF to maximum levels at 4 h after treatment (Steinmetz et al., 2000). Moreover, authors decided to clarify whether the actions of GHRH-RP, p75-92NH2 or GHRH on the stimulation of SCF mRNA levels might be mediated by growth hormone. The results obtained showed that the treatment of Sertoli cells with growth hormone did not have significant effects on the expression of SCF, and it was suggested that the actions of GHRH could be mediated by its receptor present in Sertoli cells and not by the growth hormone (Gallego et al., 2005). These findings are also supported by the study of Martínez-Moreno and colleagues showing that GHRH treatment stimulated the proliferative activity in chicken testes (Martinez-Moreno et al., 2014). The insulin-like growth factor-1 (IGF-1), an intraovarian growth factor associated with development of follicles (Silva et al., 2009), up-regulated the expression of SCF and c-KIT in the ovary (Yao et al., 2014). However, there are no other studies describing the effects of other growth factors modulating the expression levels of SCF/c-KIT system in ovarian cells. Although ghrelin functions are mainly linked with the physiology of the gastrointestinal tract, the expression of ghrelin, as well as its receptor, has been reported in the testis, which indicates that ghrelin acts directly at the gonadal level (Barreiro et al., 2004; Gaytan et al., 2004; Ishikawa et al., 2007; Tena-Sempere et al., 2002). Ghrelin is mainly located in Leydig cells (Barreiro et al., 2004; Gaytan et al., 2004; Ishikawa et al., 2007; TenaSempere et al., 2002), and an intratesticular injection of ghrelin decreased the proliferative activity of immature Leydig cells (Barreiro et al., 2004). Ghrelin treatment also diminished the expression of SCF in seminiferous tubules cultured in vitro, an effect that was visible at different developmental stages (Barreiro et al., 2004). Moreover, it was described that the expression of ghrelin by Leydig cells in human testis was inversely correlated with the serum concentration of testosterone (Ishikawa et al., 2007). Taking into account that ghrelin decreases the expression of SCF and that c-KIT activity seems to be related with the production of testosterone by Leydig cells (Rothschild et al., 2003), further investigation is warranted to clarify whether the effects of ghrelin in testicular cells are mediated by the SCF/c-KIT system. Please cite this article in press as: Figueira MI, et al. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem (2014), http://dx.doi.org/10.1016/j.proghi.2014.09.001


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6. Conclusion The regulation of SCF/c-KIT expression in male and female gonads results from a complex orchestration of many hormones and involves multiple regulatory pathways (Fig. 2). Gonadotropic and steroid hormones seem to dictate the expression levels of SCF/c-KIT system both in the testis and ovary. Moreover, abnormal levels of estrogens and androgens, respectively, in men and women, deregulate the expression of SCF and c-KIT, which may be linked with the etiology of infertility. Finally, considering the important roles of SCF and c-KIT in male and female physiology, a complete disclosure of the hormonal factors that modulate their expression levels in cells and tissues will be a critical issue for a better understanding and treatment of human infertility. References Allard EK, Blanchard KT, Boekelheide K. Exogenous stem cell factor (SCF) compensates for altered endogenous SCF expression in 2,5-hexanedione-induced testicular atrophy in rats. Biol Reprod 1996;55:185–93. Anjum S, Krishna A, Sridaran R, Tsutsui K. Localization of gonadotropin-releasing hormone (GnRH), gonadotropin-inhibitory hormone (GnIH), kisspeptin and GnRH receptor and their possible roles in testicular activities from birth to senescence in mice. J Exp Zool A Ecol Genet Physiol 2012;317:630–44. Barreiro ML, Gaytan F, Castellano JM, Suominen JS, Roa J, Gaytan M, et al. Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 2004;145:4825–34. Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler C, et al. The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev Suppl 1993:125–37. Bhattacharya I, Pradhan BS, Sarda K, Gautam M, Basu S, Majumdar SS. A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats. Am J Physiol Endocrinol Metab 2012;303:E886–98. Bialas M, Borczynska A, Rozwadowska N, Fiszer D, Kosicki W, Jedrzejczak P, et al. SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis. Andrologia 2010;42:83–91. Blanchard KT, Lee J, Boekelheide K. Leuprolide, a gonadotropin-releasing hormone agonist, reestablishes spermatogenesis after 2,5-hexanedione-induced irreversible testicular injury in the rat, resulting in normalized stem cell factor expression. Endocrinology 1998;139:236–44. Blume-Jensen P, Claesson-Welsh L, Siegbahn A, Zsebo KM, Westermark B, Heldin CH. Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J 1991;10:4121–8. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411:355–65. Blume-Jensen P, Jiang G, Hyman R, Lee KF, O’gorman S, Hunter T. Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3’-kinase is essential for male fertility. Nat Genet 2000;24:157–62. Boekelheide K. Rat testis during 2,5-hexanedione intoxication and recovery. I. Dose response and the reversibility of germ cell loss. Toxicol Appl Pharmacol 1988;92:18–27. Bokemeyer C, Kuczyk MA, Dunn T, Serth J, Hartmann K, Jonasson J, Pietsch T, Jonas U, Schmoll HJ. Expression of stem-cell factor and its receptor c-kit protein in normal testicular tissue and malignant germ-cell tumours. J Cancer Res Clin Oncol 1996;122:301–6. Breyer PR, Rothrock JK, Beaudry N, Pescovitz OH. A novel peptide from the growth hormone releasing hormone gene stimulates Sertoli cell activity. Endocrinology 1996;137:2159–62. Broudy VC, Lin NL, Liles WC, Corey SJ, O’laughlin B, Mou S, et al. Signaling via Src family kinases is required for normal internalization of the receptor c-Kit. Blood 1999;94:1979–86. Bull P, Morales P, Huyser C, Socias T, Castellon EA. Expression of GnRH receptor in mouse and rat testicular germ cells. Mol Hum Reprod 2000;6:582–6. Carranza A, Faya M, Merlo ML, Batista P, Gobello C. Effect of GnRH analogs in postnatal domestic cats. Theriogenology 2014. Carreau S, Hess RA. Oestrogens and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 2010;365:1517–35. Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, et al. Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci U S A 2004;101:6876–81.



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Review on mechanisms of dairy summer infertility and implications for hormonal intervention.

Mechanisms of beta-adrenergic receptor regulation in lungs and its implications for physiological responses.

Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog.

Hormonal regulation of vitamin D receptor levels and osteocalcin synthesis in human osteosarcoma cells.

Molecular regulation of autophagy and its implications for metabolic diseases.

The regulation of the autophagic network and its implications for human disease.

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

CD40 and its ligand, an essential ligand-receptor pair for thymus-dependent B-cell activation.

The hormonal composition of follicular fluid and its implications for ovarian cancer pathogenesis.

Regulation of immune responses by the neonatal fc receptor and its therapeutic implications.

The origins of genetic variation between individual human oocytes and embryos: implications for infertility.

Evolution of gonadotropin-inhibitory hormone receptor and its ligand.

Hormonal regulation of formiminotransferase.

Quantification of ligand bias for clinically relevant β2-adrenergic receptor ligands: implications for drug taxonomy.

Transport regulation of two-dimensional receptor-ligand association.

Undescended testis and infertility-Is hormonal therapy indicated?

Ligand regulation of a constitutively dimeric EGF receptor.

Immunological and hormonal effects of exercise: implications for cancer cachexia.

Nuclear lamina remodelling and its implications for human disease.

Ligand recognition by purified human mannose receptor.

A model for the C5a receptor and for its interaction with the ligand [corrected].

Clomiphene treatment for women with unexplained infertility: placebo-controlled study of hormonal responses and conception rates.

EGF receptor down-regulation attenuates ligand-induced second messenger formation.

Network identification of hormonal regulation.