Endocrinology Vol. 145, No. 11 4822-4824
Copyright © 2004 by The Endocrine Society
Divide and Differentiate: Ghrelin Instructs the Leydig Cells
T. Rajendra Kumar
University of Kansas Medical Center Department of Molecular and Integrative Physiology Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: Dr. T. Rajendra Kumar, University of Kansas Medical Center, Department of Molecular and Integrative Physiology, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. E-mail: tkumar{at}kumc.edu.
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Introduction
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An interesting report by Barreiro et al. (1) in this issue of Endocrinology provides compelling evidence for a novel testicular role of ghrelin, a recently discovered peptide expressed mainly in the stomach, in regulating the Leydig cell proliferation.
The Leydig cells are important somatic cells within the testis interstitium. During testis development, they originate and differentiate in distinct phases (i.e. fetal and adult) that are regulated by a plethora of autocrine, paracrine, and endocrine growth factors (2, 3). In the pubertal and postpubertal testis, three morphologically and functionally well-defined populations of adult type Leydig cells arise sequentially. Although much debated in the past, it is now well established that the fetal Leydig cells arise independently of gonadotropin influence, proliferate, produce testosterone, and persist in small numbers in the adult (4).
More complex is the regulation of proliferation and differentiation of the adult-type Leydig cells. During pubertal testis development, a small subset of mesenchymal progenitor cells initially gives rise to precursor cells, which subsequently undergo a developmental transition, first into immature and finally mature Leydig cells (3, 5, 6). Each of these critical steps is biochemically characterized by the acquisition of responsiveness to LH, which is the principal steroidogenic trophic hormone released from the pituitary (3, 5, 6). Typical of many developmentally regulated cell lineages that are important for organismal physiology, adult Leydig cells also initially proliferate rapidly, undergo progressive cessation of cell cycle, and terminally differentiate to produce testosterone. These developmental events must be coordinated tightly; aberrations in any of these steps may cause infertility or uncontrolled proliferation leading to Leydig cell hyperplasia and tumor formation (7, 8). The mechanisms of the Leydig cell proliferation and differentiation and the role of LH in regulating these events, directly or in concert with other factors, are poorly understood.
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Ghrelin, a Multifunctional Peptide
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Ghrelin, a 28-amino-acid peptide, was originally discovered as a ligand that binds to the GH secretagogue receptor; it acts centrally to regulate GH secretion and food intake (9, 10, 11, 12). Subsequent studies have revealed more widespread expression and action of ghrelin in many peripheral tissues, including the testis (12, 13). Earlier studies by the Tena-Sempere group (14, 15, 16, 17) have identified that, within the testis, ghrelin is mainly expressed in the Leydig cells and regulated by LH. At least two ghrelin receptors (seven transmembrane spanning and G protein-coupled), generated by alternate splicing of a single gene, are localized to both the Sertoli and Leydig cells (18, 19, 20); however, delineation of a local role of ghrelin within the testis and the downstream signaling pathways activated by ghrelin has not yet occurred.
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Proliferative and Antiproliferative Effects of Ghrelin
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Among many of the peripheral actions attributed to ghrelin, its opposing effects on cell cycle are the most prominent. Although in vitro a growth-promoting effect of ghrelin has been observed in some cases, its growth-inhibiting activity was observed in several thyroid, lung, and breast cancer cell lines (12, 21). Additional studies in the testis identified an inverse correlation of ghrelin expression with the developmental status of the adult Leydig cell populations. Thus, the expression of ghrelin is almost undetectable in poorly differentiated precursor and immature adult Leydig cell populations; but it is abundantly expressed in the well- differentiated mature Leydig cells. Similarly, when the adult mature Leydig cell population was selectively eliminated by the potent cytotoxic drug, ethylene dimethane sulfonate (EDS), expression of ghrelin was undetectable. After the EDS treatment, during a 3-wk recovery period, when the remaining and unaffected precursor Leydig cells undergo rapid proliferation and eventually give rise to a new population of terminally differentiated Leydig cells, ghrelin was again readily detectable (15, 16, 19, 20). Most importantly, expression of ghrelin during normal Leydig cell development also parallels that in transformed human Leydig cells; poorly differentiated tumor cells demonstrate undetectable expression, whereas differentiated tumors cells express significant amounts of ghrelin (19, 20). Although mostly indirect, all of these studies provoke the hypothesis that ghrelin may function as an autocrine-regulator of immature Leydig cell proliferation. This is the goal for most of the studies reported by Barreiro et al. (1) in this issue of Endocrinology.
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Autocrine Effects of Ghrelin in the Testis: Regulation of Proliferation of Immature Leydig Cells
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To directly test in vivo whether ghrelin acts as an antiproliferative regulator of immature Leydig cells, Barreiro et al. (1) have unilaterally injected synthetic ghrelin into pubertal and EDS-treated adult rat testis and analyzed its effects on incorporation of 5-bromo-2'-deoxyuridine labeling into the Leydig cells. In the testes of both these models, normally, the Leydig cells are in a peak proliferation phase, and therefore this analysis would permit monitoring the antiproliferative effects of ghrelin. Indeed, in both these models, ghrelin caused a significant decrease in immature Leydig cell proliferation with no effect on other mesenchymal cell types present in the interstitium. Thus, ghrelin acts as an important regulator of the immature Leydig cell population. Whether ghrelin treatment initiates the expression of mature Leydig cell markers remains to be addressed in the future. Ghrelin may also act on the Sertoli cells to regulate Leydig cell functions. Because stem cell factor (SCF) and anti-Mullerian hormone (AMH) are important Sertoli cell-derived positive and negative paracrine regulators of the Leydig cell proliferation, the authors have logically addressed the effects of ghrelin on expression of SCF and AMH in a series of experiments.
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Paracrine Effects of Ghrelin in the Testis: A Suppressor of SCF Expression
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Intercellular communication is a hallmark of the testis (22, 23). This is essential for progression of distinct cell populations in the testis and normal spermatogenesis. Previously, the EDS paradigm had identified important paracrine roles of SCF and AMH on the Leydig cells (24, 25). Similarly, the present studies indicate that, in the EDS-treated rat model, ghrelin inhibits SCF (the positive regulator) and not AMH (the negative regulator) mRNA expression in the testis. This correlates well with the cessation of proliferation of the differentiating immature Leydig cells. Furthermore, the authors have also demonstrated that, in vivo, ghrelin physiologically inhibits SCF that is FSH dependent. A similar effect was also observed in cultures of staged seminiferous tubules in vitro. The observation that ghrelin inhibits SCF, a ubiquitous growth factor, is interesting and may have broader implications for therapeutics of at least some types of cancers.
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The Intratesticular Role of Ghrelin: Significance and Future Directions
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More than 50 growth factors and peptides have been identified in the testis; however, only a few of them have been functionally well characterized (6, 26). Although ghrelin has been discovered recently in multiple species, the present and the previous findings of its antiproliferative role in the testis have become increasingly apparent. Programed developmental transitions (i.e. proliferation/apoptosis and differentiation) of the Leydig cells are critical for testosterone production and completion of spermatogenesis. In addition to a pituitary trophic regulation, existence of multiple self-regulatory signaling systems is important for finely tuning the functions of individual cell types within the testis. Thus, acquisition of the ghrelin/ghrelin-receptor signaling system by Leydig cells during distinct phases of testis development appears to regulate the proliferation and differentiation of this important steroidogenic cell type. Several questions remain: what triggers the expression of ghrelin in the mature Leydig cells, and how is it suppressed in the precursor and immature Leydig cells? How does ghrelin inhibit the Leydig cell proliferation; which cell cycle regulators are affected? Does ghrelin signal to crosstalk with AMH, IGF-I, and TGF-
, the other well-characterized locally acting growth factor signaling pathways in the testis? Which signaling pathways are downstream of ghrelin in the Sertoli cells to regulate SCF production? Some of these issues will require additional in vitro and more complex in vivo genetic approaches including RNA interference technology and tissue/cell-specific gene inactivation strategies (27, 28, 29, 30). It is hoped that these future studies will provide more information regarding the biological actions of ghrelin in the testis. Because ghrelin is a small peptide, it may be feasible to develop injectable small molecule ghrelin mimetics that could potentially aid in manipulating testosterone production and spermatogenesis.
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Footnotes
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Abbreviations: AMH, Anti-Mullerian hormone; EDS, ethylene dimethane sulfonate; SCF, stem cell factor.
Received September 1, 2004.
Accepted for publication September 1, 2004.
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References
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Introduction
Ghrelin, a Multifunctional...