Growth Factors and the Epididymis

Mechanisms of cell communication are of paramount importance in multicellular organisms, because they are responsible for the coordination of basic processes, such as growth, differentiation, immunity, motility, and transport. The most common mechanism of cell communication is that mediated by extracellular signaling molecules that bind membrane receptors of target cells and activate a host of intracellular signaling cascades that lead to specific biological responses. Signaling is classified as endocrine or paracrine on the basis of the proximity of the target cell. In endocrine signaling, target cells are located in a distant site and are influenced by signaling molecules arriving via the vasculature. In paracrine signaling, target cells are influenced by the secretion of neighboring cells. Autocrine and juxtacrine modes of secretion have also been recognized. During autocrine secretion, signaling molecules act on the cell that secreted them, and during juxtacrine secretion, plasma membrane molecules of one cell activate receptors in the membrane of a neighboring cell. In the epididymis, a "lumicrine" mechanism must also be considered in which signaling molecules reach the epididymis from another organ, the testis, via the luminal compartment, rather than the vasculature (Turner and Miller, 1997; Hinton et al, 1998). In recent years, testicular factors and lumicrine signaling in general have been recognized as important contributors to the physiology of the epididymis (Hinton et al, 2003). Growth factors are a group of polypeptides that promote cell division and differentiation, usually in a paracrine or endocrine fashion. They are grouped into families on the basis of their structure, with most of the families containing several members. They act by activating cell membrane receptors with tyrosine kinase (RTK) activity or, less frequently, receptors with serine/threonine kinase activity. The mechanism of RTK activation following binding of a growth factor involves dimerization and autophosphorylation of tyrosine residues, which induces the receptor to trigger the activation of mitogen-activated protein kinases (MAPKs) (Schlessinger, 2000). There are 3 well-known groups of MAPKs: extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 kinases (Johnson and Lapadat, 2002). These are highly conserved enzymes that share the common property of being activated by 3-tier phosphorylation cascades in response to a variety of stimuli. The ERK pathway is preferentially stimulated by growth factors and phorbol esters, whereas the JNK and p38 pathways are usually activated by ionizing radiation, osmotic shock, and other stress stimuli. RTK stimulation by growth factors is usually associated with activation of ERKs, particularly ERK1 and ERK2. These are highly similar serine/threonine kinases with sizes of 44 and 42 kd, respectively, that are themselves activated by double phosphorylation on tyrosine and threonine residues. The most important intracellular signaling cascade for ERK activation involves the activation of Ras, a membrane-bound GTP-binding protein (Figure 1). Similar to other GTP-binding proteins, Ras is activated by exchanging its bound GDP for GTP. This initiates a cascade of intracellular phosphorylation events in which upstream kinases phosporylate (ie, activate) downstream kinases according to the following sequence: Raf (also known as MEK kinase, MEKK, or MAPKKK), MEK (or MAPKK kinase), and ERK (also known as MAPK; Wilkinson and Millar, 2000). A host of tissue-specific adapters and scaffolds are also required for the activation of this cascade, but will not be discussed here because they are beyond the scope of this review (Roux and Blenis, 2004). Phosphorylated ERKs play a central role in growth factor signaling because they phosphorylate a variety of membrane and cytoskeletal proteins, downstream kinases, and, most importantly, transcription factors, such as Elk-1 and the proto-oncogene product c-myc, that ultimately mediate specific cell responses (Schaeffer and Weber, 1999). . Intracellular signaling by growth factors. Growth factors mediate their actions by stimulating mitogen-activated protein kinases (MAPKs). These intracellular kinases are activated by phosphorylation and are usually classified into 3 groups: extracellular signal-regulated kinases (ERKs) 1 and 2, c-Jun amino-terminal kinases (JNK), and p38 kinases. The effects of growth factors are generally mediated by stimulation of the ERK1/2 pathway, the central aspects of which are depicted here (see text for references). Growth factors attach to cell membrane receptors known as receptor tyrosine kinases (RTKs), which upon growth factor binding, dimerize and autophosphorylate tyrosine residues. These activated RTKs induce Ras to undergo activation by exchanging its bound GDP for GTP. Activated Ras promotes Raf phosphorylation in the vicinity of the cell membrane. There are actually 3 known Rafs, collectively referred to as "Raf" in the figure. Raf phosphorylates MEK (either MEK1 or MEK2), which are referred to here as "MEK." MEK activates ERK by phosphorylation of tyrosine and threonine residues. There are also 2 known ERKs, ERK1 and ERK2, referred as "ERK" in the figure. Activated ERKs accumulate in the nucleus and induce the phoshorylation of numerous substrates, including transcription factors, which promote the expression of specific genes. (Parts of the Raf-MEK-ERK pathway are also known by a MEK/MAPK terminology [see text].) In recent years, a number of investigators have explored the role of growth factors in the physiology of the epididymis and the male reproductive system in general. Epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) have received the most attention, but other growth factors have also been studied, and a concise review seems appropriate at this time because more-rapid advances are being made in the field. We have also included here references to research demonstrating the presence of growth factors in the testis, given the fact that substances secreted by the testis may influence the epididymis in a lumicrine fashion. Finally, we illustrate mRNA expression data for selected growth factors and growth factor receptors from the recently published mouse epididymal transcriptome (MET) database (available at: http:www.mrg.genetics.washington.edu), which details gene expression of all mouse epididymal segments (Johnston et al, 2005). In this study, which is a collaboration between our laboratory and those of others, mRNA expression in all 10 segments of the mouse epididymis was analyzed using Affimetrix gene chips (Figure 2). . Expression of Egf and Efgr in the epididymal segments of the mouse. Expression data were obtained from a microarray analysis originally performed by our laboratory (Johnston et al, 2005). Briefly, epididymides were obtained from anesthetized C57BL/6 mice and segments immediately dissected. Similar segments from 5 animals were pooled, and mRNA was obtained using standard phenol/guanidine isothiocyanate extraction. mRNA was then used as a template to generate biotin-labeled cRNA. Gene expression was evaluated as the hybridization strength of fragmented cRNA to MOE430A and MOE430B Affimetrix gene arrays. The figure shows hybridization signals for Egf (open bars) and Egfr (black bars) in all 10 segments. Each data point corresponds to the average hybridization signal obtained from 4-8 replicates of the pooled samples described above. Segmentation of the mouse epididymis (inset). Numbers indicate the segment number as used in the main figure, segment 1 being most proximal and segment 10 being most distal. An early indication of a potential role for EGF in the male reproductive system was the observation that the removal of the submaxillar gland—an organ rich in EGF—led to a marked decrease of epididymal sperm cells in adult mice without affecting the levels of testosterone or follicle-stimulating hormone (Tsutsumi et al, 1986). Moreover, changes were reversed by administration of EGF, confirming that the decrease in epididymal sperm cells was caused by EGF deprivation. It has been reported, however, that this effect is much smaller than originally claimed (Russell et al, 1990). EGF is a 53-amino acid, 6-kd protein that was originally discovered during the observation that mice submaxillary gland extracts accelerate the development of newborn animals (Cohen, 1962; Carpenter and Cohen, 1990). Over the years, EGF has proven to be a growth factor that stimulates not only the proliferation of epithelial cells, but also a number of other cell types of both ectodermic and mesodermic origin. The effect of EGF on spermatogenesis prompted studies that have demonstrated the presence of both EGF (Byyny et al, 1972; Elson et al, 1984) and EGF receptors (EGFRs) (Suarez-Quian et al, 1989; Suarez-Quian and Niklinski, 1990; Foresta et al, 1991) in the testes of different species, including humans, mice, and rats. Moreover, studies in mice have demonstrated the ability of testicular extracts to displace 125I-EGF from its receptor in a manner indistinguishable from that of submaxillary gland extracts, which suggests the presence of the canonical, 6-kd form of EGF in the testis (Radhakrishnan et al, 1992). Studies involving nonhuman primates confirmed the presence of the EGF receptor in the testis and demonstrated that this receptor is also present in the epididymis and the vas deferens (Radhakrishnan and Suarez-Quian, 1992). The EGFR is located in both the basolateral and apical borders of epididymal epithelial cells. Moreover, the luminal staining showed vesicular images consistent with a pattern of receptor-mediated endocytosis. Further immunohistochemical studies on C3H mice showed that the intracellular localization of the EGF receptor varies along the length of the epididymis: in the caput, staining was intense and evenly distributed in the cytoplasm of principal cells, whereas in the corpus and cauda, staining was limited to the apical cytoplasm (Suarez-Quian et al, 1994). The presence of a growth factor in a tissue does not necessarily mean that the growth factor is synthesized there. Other investigations have shown that the testis is, in fact, a source of EGF (Radhakrishnan et al, 1992). In these studies, the presence of the intracellular EGF precursor (EGFp) in the mouse testis was investigated by Western blotting and immunohistochemistry. EGFp is a 140-kd integral-membrane protein from which the 6-kd, active EGF is released by proteolytic cleavage and whose presence in a given tissue strongly suggest that EGF is locally synthesized. In contrast to EGF, which was found to be widely distributed in the testis, including in Sertoli cells, EGFp was restricted to germ cells, suggesting a paracrine signaling mechanism. The presence in the testis of EGF, which is usually a paracrine-acting growth factor, is important for epididymal physiology, given the potential lumicrine influence of testicular factors on the epididymis (Fawcett and Hoffer, 1979; Nicander et al, 1983; Hinton et al, 1998; Hinton et al, 2003). Apart from a single report of putative EGF in the human epididymis (Elson et al, 1984), the presence of EGF in the epididymis has not been determined. In this regard, it is interesting that data from MET database demonstrate that Egf is expressed in the epididymis in a segment-specific manner (Figure 2). Thus, it is possible that, in addition to the lumicrine model, cells of the epididymal interstitium (eg, macrophages and fibroblasts) synthesize and secrete EGF to communicate with cells in the adjacent tubule epithelium. Interestingly, expression of Egf is relatively low in the most proximal segments of the caput epididymis, but increases approximately 4-fold by segment 7, which comprises most of the corpus region. In this part of the duct, EGF from the testis may no longer be available to the epithelium, and it can be hypothesized that the epithelial cells may depend more on local, paracrine-acting EGF. It has been reported in the MET databases that expression of the gene encoding the EGFR is relatively constant throughout the epididymis (Figure 2); thus, if the epididymal expression of Egf and Egfr are confirmed by protein presence, the data will be consistent with the concept that lumicrine EGF is important in the proximal caput of the mouse epididymis, and that paracrine EGF is more important in more distal segments, especially the corpus epididymis. Whether this hypothesis is true in the mouse or other species is yet to be determined, but these initial gene expression data suggest that EGF plays a meaningful role in the segmental regulation of the epididymal tubule. Of note, the MET database shows that Cd97, a member of the EGF 7-span transmembrane receptor family, is up to 10-fold more highly expressed than Egfr in the mouse epididymis, especially in the cauda segments (data not shown). CD97 has an extended extracellular region with several N-terminal EGF-like domains that mediate binding to its ligand (Jaspers et al, 2001). The role of CD97 in cell signaling in macrophages and dendritic cells has previously been studied, and it will be interesting to determine whether these cells are playing a regulatory role within the segment(s) in which they reside. Fibroblast growth factors (FGFs) are a large family (>20 members) of related polypeptides whose function, in addition to promoting cell growth, includes the regulation of cell motility, differentiation, chemotaxis, and apoptosis (Böttcher and Niehrs, 2005). They are found in a wide variety of species and play an important role in the ontogeny of both vertebrates and invertebrates, although their biological role in adult tissues is much less understood. Vertebrate FGFs are single-chain polypeptides with an approximate size of 17–34 kd that share a common, highly conserved central core domain of 120 amino acids and exhibit preferential binding to specific FGF receptors (FGFRs; Ortniz and Itoh, 2001). Similar to other growth factor receptors, FGFRs are membrane proteins with extracellular ligand-binding domains and intracellular tyrosine kinase domains that promote autophosphorylation and dimerization. Four different types of FGFRs have been described (FGFR 1–4) that correspond to the products of 4 highly related genes (Fgfr-1–4). Alternative splicing of Fgfr transcripts, particularly types 1–3, generates a variety of receptor isoforms that are differentially expressed along the mouse epididymis (MET database). Signaling by FGFs usually proceeds via the classical Ras/ERK cascade (see above), although in some tissues, signaling occurs via activation of PLCγ (Böttcher and Niehrs, 2005). In the latter case, activated FGFRs bind PLCγ, which hydrolyzes phosphatidylinositol-4,5-disphosphate into diacylglycerol and inositol 1,4,5-trisphosphate (IP3). This provides a link to signaling cascades mediated by Ca2+, given the effect of on intracellular The FGF was discovered as a that stimulates the proliferation of cells and It was to be from FGF from the Similar to EGF, has been shown to be present in the testis et al, et al, et al, studies have shown that the testis a factor that stimulates the growth of et al, and that of and human epididymides exhibit activity et al, MET data suggest the of expression of both and along the length of the epididymis. The 2 most highly expressed FGF and pattern of with being more highly expressed in caput segments and in segment of the cauda (Figure the is the most by and in the cauda epididymis (Figure . Expression of (open bars) and (black bars) in the epididymal segments of the mouse. Expression data were obtained as described in . Expression of (open (black and bars) in the epididymal segments of the mouse. Expression data were obtained as described in the biological of these is not yet reported by Hinton et suggest a lumicrine of for Hinton and investigated the influence of on the activity of epididymal which the epididymal of testicular factors et al, 1998). is to sperm cells from stress by levels of which the of protein to et showed that the levels of and activity that are in the initial segment of the epididymis and that low activity the of stimulation by a testicular factor or factors. Interestingly, activity can be with testis but not with EGF. these an of the regulation of an important epididymal by testicular factors, and suggest as a In a related study, the presence of FGFRs in cells from the initial segment of the epididymis was investigated by and et al, 2003). 4 types of FGFRs were found to be expressed in the initial with principal cells only these suggest a influence of the on the epididymis. was as a factor that increases the of et al, and was found to be a specific for vascular endothelial cells et al, 1989; et al, plays a role in during and because development the of and and 2005). During the years, a of biological for have been in to the and of of these are regulation of and of apoptosis et al, 2004). The family comprises and and growth The most expressed member is a acid, usually referred to as In can be to generate isoforms with and, more and amino is the most expressed in the mouse epididymis (Figure especially in the proximal segments. of the family with different with 3 different and et al, 2003). These receptors are also known as (or for the and Of the gene encoding is expressed more than more than the other in the mouse epididymis, whereas the gene encoding has the expression (MET database). . Expression of (black (open and bars) in the epididymal segments of the mouse. Expression data were obtained as described in The of and its receptors, as as the effect of on epididymal have been recently investigated in the human epididymis et al, 1998). analysis has shown that as as mRNA for the receptors and are present in human epididymis The expression of protein was confirmed by with and its receptors were by in of human tissue was to cells and in endothelial cells from was in the vascular and in some cells. for in the human epididymis was with the of some in but was strongly in and cells of the distal part of the In the has been by in hybridization in endothelial cells of throughout the epididymis et al, 1998). Finally, of human epididymides with has shown an number of and intracellular which suggests that several aspects of epididymal may be influenced by via activation of the A potential role of in the reproductive system of the mouse has been explored in mice the mouse et al, 1998). These mice spermatogenesis and a epididymis and, as a are not The epididymides of these animals are and has other that the in the caput epididymis is in with whereas the cauda shows a epithelium. These are of those in mice with a proximal although this was not in the by et Interestingly, the of epithelial expression of both and as as an number of and et al, 1998). Whether this on the role of in the epididymis, or on a related of the studies with an the amino of that in the testes is to and Sertoli cells et al, from Sertoli cells reach the epididymis via the lumicrine and influence the activity of the epididymal epithelium. This be consistent with the that testicular factors, including play an important role in the physiology of the epididymis. it may be the that be playing a paracrine role in the early segments of the mouse epididymis because expression is relatively in this region (Figure This be consistent with the of et that showed of to cells in the human epididymal compartment, including growth factor is a of that plays a central role in the development of and also promotes growth and 2003). It is also known as because the of a 140-kd has been in the mouse and testis and epididymis by in hybridization and blotting et al, In the adult mouse epididymis, in hybridization signals were in the of the corpus and in some principal cells of the The however, although in the mouse testis is in the epididymis, as by in This is also consistent with the gene expression data available in the MET Interestingly, obtained from mouse testis and epididymis showed biological activity for in from The of these is not yet although it has been that epididymal may play a role in sperm et al, This is by the large number of signaling in which and other are not only in but other cell types as and 2003). a role of in the development of mouse testis and epididymis has also been on the basis of in expression of and receptors et al, 1999). growth factor and the receptor are also present in the and mouse epididymis of both and animals et al, 2004). The family comprises a of and by a of 4 and which bind to different receptors, and and exhibit activity on tissue cells et al, 2002). In the adult principal cells intense along the length of the epididymis for and et have also shown that, at approximately of the epididymis of mice exhibit of a in size and an epithelium. and receptor mice immediately and no epididymal These have on the basis of suggesting that may influence in the epididymis of the et al, that may be in the regulation of the secretion of this and, other proteins by the of the epididymis. The growth family comprises isoforms of and They are secreted by and cells and are of cells in They have been shown to influence processes, such as growth, differentiation, and apoptosis and 2003). It is known that is present in the testis et al, and both et al, and Sertoli cells et al, less is however, the role of in the epididymis. It has been recently demonstrated that and receptor are both present in the epithelial cells of the caput and corpus of the epididymis of the et al, 1999). was to apical cells, the receptor was restricted to principal but in both a of cells was in the caput than in other confirmed expression in the epididymis et al, 1999). These prompted the hypothesis that may have a paracrine role in the epididymis, in to the effects of and Similar studies in the epididymis have shown that localization is rather than but to on the other is epithelial and restricted to the corpus et al, 1998). analysis by blotting confirmed these and showed that the expression of mRNA in the corpus is than that in the caput and Studies involving also the of an between and and 2000). In these mRNA expression by increases in the approximately 10-fold to and less in the caput and importantly, these changes are reversed by which is is in the cauda, and expression increases in the caput, but is much less in the corpus and does not in the In the according to the MET the gene encoding is the form most especially in segment The presence of growth factor in the epididymis was originally reported by et and are proteins kd in related to that play important and autocrine in and tissues and Cohen, 2000). These biological effects are by binding proteins that the of and, in some as A of the of in the epididymis has shown that, at early is to epithelial cells and particularly in the cauda, is less in the epithelium, and more intense in cells. Expression of the genes encoding and is not than levels in the adult mouse epididymis (MET database). Studies of the of an however, that this protein is present in the of the human epididymis et al, 2000). The of these is but it is possible that in some species, may have a role in the epididymis. It has recently been reported that is by the mouse epididymis et al, 2002). shows that the epididymis mRNA at relatively approximately of the levels in the of was confirmed by epididymal tissue for in the presence and the of the protein of in the as by were by this Interestingly, was of testosterone and In hybridization shows that is to be by cells. is a secreted by the and the adult that is for because it stimulates to and 1992). the is the most important source of during other tissues, such as the and reproductive are also and 1999). Interestingly, the pattern of secretion by the epididymis that of the is an initial by a of which with the response of other that exhibit a more response et al, 2002). it has been that the epididymis may be a source of et al, 2002). This has recently been by the of the presence of in the epididymis et al, which is a transcription factor that activates several genes in including The gene encoding is not in the MET but receptor is expressed in all 10 epididymal segments shown). Finally, it has been that growth factor may be a of the by sperm cells during epididymal is a and found in a variety of tissues et al, 2005). in that as sperm cells from the caput to the cauda epididymis, they a more of a receptor for et al, 2002). This has been with involving mice, in which was by in the mouse distal corpus and cauda, but not in the caput, and with the fact that initiates of not sperm cells et al, 1994). Moreover, et have shown that the of caput sperm cells is in by the presence of These investigators that may be important in the of sperm cell in the epididymis because these cells are to of as they to the cauda, and in because they may be more to this to the in The fact that sperm cells are in in the cauda epididymis of most species suggest that this is of a more (Turner and et al, The studies here have to a of related to possible of growth factors in the epididymis. They are in that they have the presence and of different growth factors in the epididymis of different species and have potential biological for these for the most these studies have not beyond Moreover, given the of the data different species and growth factors, and the fact that studies have on a single it has not been possible to a for the of A consistent data pattern has not and in it is not that in one species can be to the role of growth factors in the epididymis be from localization studies more and studies, such as those performed by et and Hinton and et al, 1998; et al, are In this an important of the of growth factors are the intracellular signaling cascades activated by growth factors in the and the we for that the of the and families are expressed in the mouse epididymis et al, et al, much more these and other intracellular is required to the biological role of growth factors on the epididymis. of the physiology of the epididymis that the epididymal tubule is within segments (Turner et al, et al, 2005). investigations can be that the role of growth factors within those segments. are in different segments to paracrine or lumicrine In the involving and it was demonstrated that and expression have segment-specific and depend on and testicular factors and et al, 2001). It is also that the of ERK activation may play a role in the of the epididymis. ERK activation is a specific and of kinases and other molecules (Schaeffer and Weber, for and with other signaling such as cascades et al, 2002). investigations are to the role of growth factors in the epididymis, the has to possible for the one a of has been in of the that growth factors play a role in epididymal These be the aspects of growth factors at the and the other the concept of epididymal provides a to our of this the mouse epididymal transcriptome epididymal segments, or in with adjacent segments, form of gene expression are to be differentially influenced by selected growth factors. The of these may to be for the biological effect of growth factors on the epididymis, and for our of the ability of the epididymis to promote sperm We for of the