Endocrinology Vol. 147, No. 8 3662-3665
Copyright © 2006 by The Endocrine Society
Toward Understanding the Endocrine Regulation of Gonadal Somatic Cells
Matti Poutanen
Department of Physiology, Institute of Biomedicine, University of Turku, FIN-20014 Turku, Finland
Address all correspondence and requests for reprints to: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, FIN-20014 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.
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Introduction
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In addition to the sex-determining transcription factors, recent studies have provided novel information for the crucial role of several paracrine and endocrine signaling systems for the differentiation and proper functioning of steroidogenic cells in the mammalian gonads. Furthermore, the regulation of steroidogenic cell fate and steroid biosynthetic enzymes seems to be coupled. In this issue of Endocrinology, the work by Couse et al. (1), together with other recent findings, demonstrates that estrogen receptor 1 (ESR1, ER
) action inhibits the Leydig-cell appearance in the ovarian stroma. In the absence of ESR1, LH-regulated ectopic expression of hydroxysteroid (17-ß) dehydrogenase 3 (Hsd17b3), a typical feature for testicular Leydig cells, appears in the ovary. The work presented by Couse et al. (1) is an essential piece of information toward understanding the development and differentiation of gonadal somatic cells by endocrine factors, some of which is summarized below.
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LH
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One crucial factor regulating gonadal somatic and germ cell differentiation and steroidogenesis is gonadotropin stimulation. Full inactivating mutations of the LH/choriogonadotropin receptor (LHCGR) in humans result in male pseudohermaphroditism, low testosterone and high LH levels, total lack of responsiveness to LH/human chorionic gonadotropin challenge, and the absence of secondary male sex characteristics (for review see Ref. 2). In contrast to humans, in the mouse, fetal testosterone production is independent of LHCGR, but adult-type Leydig cells do not differentiate in Lhcgr knockout mice (LuRKO mice) (3). Thus, a lack of the adult-type Leydig cells is the primary cause for the absence of the pubertal testosterone production in LuRKO mice, not the lack of LH stimulus in the Leydig cells. It remains to be studied whether the differentiation of adult-type Leydig cells could be rescued in the LuRKO mice by postnatal androgen treatment or whether the direct action of LH is mandatory for the adult-type Leydig cells to develop in the testis. Whereas the differentiation of adult-type Leydig cells is directly or indirectly dependent on LH action, ovaries of the LHCGR-deficient mice have both of the ovarian steroidogenic cell types essential for sex steroid production: the granulosa and theca cells (3). However, no estradiol is produced in the absence of LH signaling, and folliculogenesis is blocked at the early antral stage (4).
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Wingless-related mouse mammary tumor virus integration site 4 (Wnt4)
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Among other functions in organogenesis and female reproduction, WNT4 is essential for ovarian cell differentiation and function. Wnt4 is a member of the Wnt gene family, which encodes secreted growth and differentiation factors acting through Frizzleds and their coreceptors. In many situations Wnt signaling activates the so-called canonical Wnt signal transduction pathway that is initiated by ligand binding and leads to accumulation of ß-catenin in the cytosol. In normal conditions, ß-catenin binds to the Tcf transcription factor and the protein complex translocates to the nucleus and activates target genes. WNT4, besides being important in several other organs, is also essential for the development of the ovary and its function. The Wnt4 newborn KO mice have masculinized ovaries that produce testosterone, a property that is typical for the Leydig cells (5, 6), and the ovaries also present with sex cords similar to those in the male. Moreover, the Wnt4-deficient females have Wolffian ducts developed normally by androgens in male embryos. These finding are consistent with the idea that WNT4 inhibits ovarian testosterone production during the fetal period and promotes ovarian development. However, it has been shown that WNT4 is also involved in early testis development, because Sertoli cell differentiation is compromised in the Wnt4 KO testes (7).
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Sex steroids
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Balanced estrogen and androgen action is essential for proper sexual differentiation in both genders, and the dysfunctions caused by enhanced estrogen exposure in the male reproductive tract have generated much interest in this topic. It is suggested that many of the adverse effects on the testis caused by exposure to estrogens result from a combination of high estrogen and low androgen action, whereas high estrogen or low androgen action alone are unable to induce similar changes (for review see Ref. 8). A transgenic mouse model (AROM+) expressing human cytochrome P450, family 19, subfamily a, polypeptide 1 (Cyp19a1, P450 aromatase; converting androgens to estrogens) has been generated to mimic the situation with high estrogen/low androgen action (9). AROM+ mice present with Leydig cell hypertrophy and hyperplasia and a marked increase in the number of activated macrophages in the testicular interstitium, resulting in disrupted spermatogenesis associated with increased fibrosis in the testicular interstitium, a phenocopy of inflammation-associated infertility in men (10). A wealth of data suggests that testicular Leydig cells are a target for direct actions of high levels of estrogens. Among other effects, evidently estrogens cause cryptorchidism (undescended testis) in rodents. The mechanisms of the estrogen-induced cryptorchidism are one of the best characterized estrogen-mediated dysfunctions in the male reproductive tract (11). It has been shown to be associated with the down-regulation of the gene encoding the insulin-like 3 (Insl3), a Leydig-cell-derived hormone involved in gubernaculum development and responsible for transabdominal testis descent (12).
Similar to the finding in AROM+ males, testicular atrophy has also been detected in the Esr1 knockout (ERKO) males. Interestingly, it appears that the phenotype is not a direct consequence of the lack of ESR1 action on testicular cells but is a secondary effect caused by back pressure of the accumulating luminal fluids not properly drained by the efferent ductules (for review see Refs. 13 and 14). Furthermore, ßERKO mice lacking functional estrogen receptor 2 (Esr2, ERß) appear not to have altered testicular function (15). Based on the current evidence, the conclusion is that lack of estrogen signaling is not crucial for Leydig cell function, whereas increased exposure to estrogens is detrimental (see Ref. 16 for review), at least in mice. Both Esr1 and Esr2 are expressed in mouse Leydig cells, whereas the Esr1 expression in human adult-type Leydig cells has not yet been fully confirmed. However, Cyp19a1 is endogenously expressed in the testis at low levels, especially in the germ cells (for review see Ref. 17). Thus, the role of intratesticular estradiol production as a local autocrine/paracrine modulator is being actively researched. This is further corroborated by the observation that aging Cyp19a1-deficient mice (aromatase KO, ArKO) mice develop postmeiotic defects in spermiogenesis, coinciding with an elevation in apoptosis and a reduction in fertility (18, 19).
Interestingly, recent data indicate an essential role for estrogen action in the differentiation and fate of both ovarian granulosa and theca cells in the mouse. The adult ßERKO mice (20, 21), lacking both nuclear estrogen receptor subtypes, present with ovary structures resembling the seminiferous tubules, identical to that observed in newborn Wnt4 KO mice. The granulosa cells seem to transdifferentiate toward a Sertoli cell phenotype (20, 21) (Fig.
). Similarly, ArKO mouse ovaries (22) present with characteristics of both testicular interstitial Leydig cells and of seminiferous tubule-like structures with Sertoli-like cells. A logical conclusion is that in the absence of ligand-mediated actions of both estrogen receptors, ESR1 and ESR2, granulosa cell commitment is partially lost, even in the ovarian environment. The possible appearance of testicular-like cell differentiation in the ovaries of LuRKO mice lacking estradiol production, but with functional ESR1 and ESR2, remains to be studied. The Esr1 KO mice (ERKO, with functional Esr2) also have a severe ovarian phenotype, in which follicles fail to mature or ovulate and form hemorrhagic cysts, leading to infertility (21, 23). This phenotype was, however, explained largely by the chronically elevated LH levels and enhanced ovarian steroidogenesis (24). These studies implicate ESR1 in most of the negative-feedback effects of estrogens on pituitary gonadotropin secretion. In this issue of Endocrinology, Couse et al. (1) provide additional evidence for a local action of estrogens in preventing the development of functional Leydig-like cells in the stromal-interstitial portion of the mouse ovary and that these actions are mediated specifically by ESR1. Recent work also supports the idea that, of the two estrogen receptors, the ESR2 plays a predominant role in follicular maturation. The data demonstrated abnormal follicular maturation, increased atresia, and early follicle exhaustion in ßERKO mice (25). Preantral follicles from ßERKO mice cultured in vitro demonstrated slower growth, decreased estradiol secretion, and reduced ovulatory capacity compared with follicles from wild-type or ERKO mice (26).
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FIG. 1. Inhibition of transdifferentiation of mouse ovaries toward testis-like structures by hormonal factors. ßERKO mice deficient in Esr1 and Esr2 and mice lacking ovarian estradiol production because of the Cyp19a1 KO show transdifferentiation of ovaries postpubertally with structures resembling seminiferous tubules containing Sertoli-like cells (Sc). These ovaries also show increased expression of Sertoli cell markers such as Sox9 and antimullerian hormone (AMH). Similar transdifferentiation is not seen in mice deficient in Esr1 or Esr2 only. However, Esr1 KO show the appearance of Leydig-like (Lc) cells in the ovarian stroma, with ectopic expression of Hsd17b3 that is regulated with LH similar to that in testicular Leydig cells. The up-regulation of another estrogen-sensitive Leydig cell gene, Insl3, remains to be studied. An ovarian transdifferentiation with sex cords similar to those in the male, testosterone (T) production, and ectopic Hsd17b3 expression has also been observed in Wnt4 KO mice at birth. Interestingly, in the Wnt4 KO mice, a reduced ovarian expression of Esr1 has also been observed. Furthermore, estradiol may inhibit Wnt4 expression in the ovary, similar to that detected recently in the uterus. Thus, evidence exists to support the molecular interactions between the Wnt pathway effectors ß-catenin (ßC) and lymphoid enhancer factor 1 (LEF1), and ESR1 and ESR2, in the mechanism of ovarian transdifferentiation. However, missing links are to be found still. The question mark (?) indicates other factors remaining to be characterized. E2, 17ß-Estradiol; gc, granulosa cell; Oc, oocyte; tc, theca cell; SOX9, SRY-box containing gene 9; AMH, anti-Mullerian hormone.
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The work by Couse et al. (1) presented in this issue of Endocrinology also demonstrated that ESR1 inhibits the ectopic expression of Hsd17b3 (an enzyme converting androstenedione to testosterone) in the ovary. In ERKO mice, the expression of this enzyme, specific for Leydig cells in adult male mice, is highly expressed in the ovaries and is regulated by LH, similarly to that expected in Leydig cells. Interestingly, similar ectopic expression of Hsd17b3 has been detected in adult ArKO (27) females and newborn Wnt4 KO females (6). In the ERKO, ArKO, and Wnt4 KO mice, the induced expression of Hsd17b3 likely explains the increased production of testosterone in the ovary. However, Hsd17b1 also seems to be expressed in Esr1 (24) and Wnt4 (6) KO mice. The mouse HSD17B1 equally well converts androstenedione to testosterone and activates estrone to estradiol. The enzyme, thus, is likely also to contribute to the testosterone production in the ERKO, ArKO, and Wnt4 KO mice (28). Because Cyp19a1 is also shown to be expressed in both Wnt4 KO (6) and ERKO mice ovaries (1), the lack of efficient conversion of testosterone to estradiol is surprising. One possible explanation would be the lack of proper electron donor systems essential for CYP19A1 activity.
The essential role for estrogens in the regulation of Hsd17b3 expression also in testis is suggested by the observation that there is an increase in Hsd17b3 expression in ERKO testis (29). This is solid evidence to support the idea that the two Leydig-cell-expressed genes, Insl3 and Hs17b3, are directly suppressed by estrogens, at least in mouse. Estrogen exposure in males represses Leydig cell Insl3 expression, whereas the lack of the inhibitory effects of estrogens induces an ectopic expression of Hsd17b3 in the ovary. It will thus be interesting to resolve the similarity of regulation of Insl3 and Hsd17b3. Is Hsd17b3 also markedly suppressed by estrogens in the testis, and is Insl3 induced in the ovaries in the absence of estrogen action or by the absence of WNT4? Notably, the data support the hypothesis that down-regulation of Hsd17b3 is one mechanisms by which estrogens directly inhibit Leydig cell testosterone biosynthesis, thereby further increasing the estrogen/androgen ratio detrimental for testicular function.
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Interaction between Wnt4 and estrogen receptors
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Surprisingly, similar ovarian phenotypes have been described for adult ERKO and ArKO and newborn Wnt4 KO mice, including testosterone biosynthesis with the ectopic expression Hsd17b3. Thus, additional studies are crucial to reveal the possible cross-talk between these signaling systems in the regulation of ovarian somatic cell fate and steroidogenesis. Potential cross-talk between Wnt/ß-catenin and estrogen signaling has been implicated in the uterus (30), for example, where estrogen has found to up-regulate Wnt4 expression. This was independent of the nuclear estrogen receptors. Furthermore, in the Wnt4 KO ovaries, a marked down-regulation of Esr1 was observed (6). It has recently been shown that ß-catenin is able to directly interact with ESR1 (31). ß-Catenin recruitment to estrogen-responsive elements and ESR1 recruitment to Tcf/Lef1 binding sites (mediating Wnt-signals) were identified in certain promoters of endogenous target genes, and both interactions were shown to be ligand dependent (31). Several recent studies have shown a cross-talk between the signaling systems of Wnt ligands and various nuclear receptor family members (for review see Ref. 32), representing the clinical potential for interfering with such interactions by pharmacological intervention. Additional studies with the genetically modified mouse models are expected to enable a better understanding of this important area of endocrinology and cell biology.
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Footnotes
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Abbreviations: ArKO, Aromatase knockout; ERKO, Esr1 knockout; Esr1, estrogen receptor 1; Hsd17b3, hydroxysteroid (17-ß) dehydrogenase 3; LHCGR, LH/choriogonadotropin receptor; LuRKO, Lhcgr knockout; Wnt4, Wingless-related mouse mammary tumor virus integration site 4.
Received May 5, 2006.
Accepted for publication May 9, 2006.
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Introduction
LH
