Receptor Biology Section (J.F.C., M.M.Y., V.R.W., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and Department of Environmental and Molecular Toxicology (J.F.C.), North Carolina State University, Raleigh, North Carolina 27695
Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, MD B3-02, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov.
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ABSTRACT |
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INTRODUCTION |
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The fact that estradiol appears to play numerous modulatory roles within all three components of the HPG axis has made it difficult to delineate the overall contribution of each site of action in reproductive function. Furthermore, the presence of both known forms of nuclear estrogen receptor (ER), ER and ERß, throughout the tissues of the HPG axis makes it equally difficult to ascertain which receptor mediates the various estrogen effects. For example, the regulatory actions of estradiol on the hypothalamus were historically considered indirect as ER remained undetectable in GnRH-secreting neurons. However, recent reports of estradiol binding and ERß-encoding transcripts in GnRH-secreting neurons of the mouse hypothalamus provide evidence that estrogen regulation of GnRH secretion may indeed be direct and mediated by ERß (5, 6, 7). Therefore, it may be anticipated that the different ER knockout (ERKO) lines, especially the ßERKO, may exhibit altered gonadotropin secretion. In the ovary, both ER forms are easily detectable but distinctly localized among the functional units of the follicle (8, 9). ER
is the predominant receptor form present in interstitial and thecal cells, which are the primary sites of LH action and androgen synthesis; ERß is localized to the granulosa cells of growing follicles, which are the primary sites of FSH action and estradiol synthesis. Therefore, it is conceivable that the intraovarian actions of estradiol may indeed differentially regulate the gonadotropin response and steroidogenic capacity of the somatic cell types in the follicle.
Previous studies toward elucidating the precise sites of estrogen action in the HPG axis and the particular ER form involved have relied upon pharmaceuticals to inhibit ER function or estradiol synthesis. However, these efforts more often led to inconclusive results as such drugs were only partially effective in blocking the enormous levels of estradiol and aromatase activity in the ovary. Furthermore, there are no ER isoform-specific agonists or antagonists that have proven effective in vivo in the ovary. Therefore, the generation of gene-targeted mice lacking one or both ER forms provides the long-awaited tools necessary to overcome these obstacles. Herein, we have thoroughly characterized the basic function of the HPG axis in the females of all three existing lines of estrogen receptor-knockout (ERKO) mice, those lacking a functional Esr1 gene (ERKO), or Esr2 gene (ßERKO), or both (
ßERKO), in anticipation that such a comparison may elucidate the contribution of each ER form in female reproductive endocrinology. Our assessment of the HPG axis in each ERKO line includes the following: 1) gene expression assays for GnRH-receptor (Gnrhr); the gonadotropin subunits, glycoprotein-hormone
-subunit (Cga), LH-ß (Lhb), FSH-ß (Fshb), and prolactin (PRL) (Prl) in the anterior pituitary; 2) plasma levels of the respective pituitary hormones, LH, FSH, and PRL; 3) gene expression assays for the components necessary for sex steroid synthesis, i.e. steroid acute regulatory protein (Star), the cytochrome P450s (Cyp11a, Cyp17, and Cyp19), 3ß-hydroxysteroid dehydrogenase (HSD)/
5-
4 isomerase (Hsd3b1), and 17ß-HSD types I and III (Hsd17b1, Hsd17b3); 4) plasma levels of the sex steroids; and 5) gene expression assays for the inhibin/activin subunits, inhibin-
(Inha), inhibin-ßA (Inhba) and inhibin-ßB (Inhbb), and follistatin (Fst) in both the ovary and anterior pituitary. In general, our findings indicate that ER
but not ERß is essential in mediating the negative-feedback effects of estradiol on the hypothalamic-pituitary (HP) axis, as only
ERKO and
ßERKO females exhibited elevated LH synthesis and secretion. The resulting hyperstimulation of the ovary in
ERKO females leads to increased Cyp17, Cyp19, and Hsd17b1 expression, which ultimately manifests as elevated levels of plasma androstenedione and estradiol. However,
ßERKO ovaries do not consistently exhibit increased Cyp17 and Cyp19 expression or estradiol synthesis despite possessing a more severe increase in plasma LH relative to the
ERKO. Most interesting was an apparent endocrine sex reversal observed in
ERKO/
ßERKO ovaries, characterized by the ectopic expression of Hsd17b3, an enzyme-encoding gene known to be specifically expressed in testes.
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RESULTS |
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The effect of ovariectomy and ER gene disruption on Prl expression in the anterior pituitary is shown in Fig. 2. As expected, ovariectomy in wild-type females resulted in a greater than 80% reduction of Prl mRNA levels in the pituitary. However, a more dramatic reduction in Prl expression was observed in both
ERKO and
ßERKO females, whereas pituitaries from ßERKO females exhibited normal Prl expression levels. Interestingly, the reduced Prl expression observed after ovariectomy or the loss of ER
was not reflected in plasma PRL levels, which were slightly reduced in each but within normal range (Table 2
). However, it should be noted that the plasma PRL levels detected in all groups were at the lower limit of detection for the assay used. Due to limited sample volume, PRL levels were not assessed in
ßERKO plasma.
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As previously reported (13) and repeated here, ERKO females exhibited elevated plasma testosterone levels relative to wild-type (30-fold), which approach the minimum level of detection (Table 2
). A more modest but significant (15-fold) increase in plasma testosterone was exhibited by
ßERKO females. However, our evaluation of steroidogenic enzyme expression in
ERKO and
ßERKO ovaries does not satisfactorily explain this enhanced capacity to synthesize testosterone, as the enzymes necessary for rapid conversion of androgen precursors to estrone or estradiol were present at normal to excess levels in each (Fig. 3
). We therefore assayed for expression of the Hsd17b3 gene, which encodes the enzyme that preferentially converts androstenedione to testosterone (see Fig. 3
). In agreement with previous reports (11), Hsd17b3 transcripts were undetectable in wild-type ovary when assayed by ribonuclease protection assay (RPA). However, Hsd17b3 expression was easily detectable in
ERKO and
ßERKO ovaries, but absent in the ßERKO. In fact, the levels of Hsd17b3 expression in the
ERKO and
ßERKO ovaries were comparable to that found in adult wild-type testis (Fig. 3
).
Effect of a GnRH Antagonist and Antiandrogen on the ERKO HPG Axis
We have previously shown that treatment of ERKO females with a GnRH antagonist effectively returns plasma LH levels to within the wild-type range and concomitantly alleviates the hemorrhagic and cystic morphology that is a hallmark of the
ERKO ovary (14). To more fully characterize the effects of such treatment in the
ERKO, we have evaluated the expression of Cga, Lhb, and Fshb in the pituitaries of similarly treated females. As expected, GnRH antagonist treatment completely erased evidence of elevated Cga and Lhb expression in the pituitary of
ERKO and ovariectomized wild-type females (Fig. 4
). Similar treatments were also effective in reducing the elevated Fshb expression that occurs after ovariectomy in wild-type females (Fig. 4
). Accordingly, plasma LH and FSH levels in GnRH antagonist-treated
ERKO and ovariectomized wild-type females were reduced to within the range of untreated intact wild-type females (Table 2
), directly correlating with the changes in gene expression. These data strongly implicate the hypothalamus as the primary site of estradiol-mediated negative feedback on gonadotropin gene expression and secretion in the rodent and definitively show this action to be dependent upon functional ER
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DISCUSSION |
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Hypothalamic-Pituitary Function in the ERKO Female
The severe phenotypes exhibited by the ERKO females, in contrast to the lack of comparable phenotypes in the ßERKO, indicate that ER
is the predominant receptor form involved in maintaining homeostasis in the female HPG axis. Certain findings in this study are congruent with the previous conclusions of others and hence require only minimal discussion. For example, our demonstration that ER
and not ERß mediates estrogen regulation of Prl gene expression in the pituitary agrees with a previous report in the
ERKO (15) and now extends these findings to the
ßERKO female. Our observation that reduced Prl expression in the pituitary after either ovariectomy or the loss of functional ER
was not reflected in plasma PRL levels (Table 2
) is also consistent with previous reports of only modest decreases in plasma PRL despite reduced pituitary content and gene expression after ovariectomy in mice (16, 17, 18). Second, it is well known that ovariectomy in female rodents promptly leads to increased LH synthesis and secretion from the pituitary and that these effects are completely prevented upon estradiol replacement, implicating the steroid as the critical ovarian-derived factor in determining plasma LH levels (1). Our data convincingly demonstrate that this pathway is dependent upon ER
, as only those mice lacking functional ER
invariably exhibited elevated levels of Cga and Lhb gene expression and plasma LH. In the wild-type rodent, it is believed the hypothalamus, rather than the pituitary, is the primary site of estrogen feedback and that ovariectomy leads to increased hypothalamic GnRH secretion, which then manifests as hypergonadotropism (1). Our findings that ovariectomy-induced hypergonadotropism was prevented by treatments with a GnRH antagonist supports this conclusion. More importantly, our demonstration that
ERKO females exhibited a comparable response to GnRH antagonist treatment indicates that the loss of ER
in the hypothalamus is similar to ovariectomy in this regard. Third, although experimental testosterone treatments have been shown to prevent ovariectomy-induced increases in Cga and Lhb expression in the female rat pituitary (19), the elevated serum androgens inherent to the
ERKO and
ßERKO females appeared to have no attenuating effect in these animals.
It is plausible that other phenotypes in the HPG axis of ERKO females would be comparable to the effects of ovariectomy in wild-type mice. This may be especially expected in the ßERKO, as the loss of both ER forms may be considered most analogous to the loss of estradiol. Indeed, Britt et al. (20) reported that female mice lacking a functional Cyp19 gene and therefore the capability to synthesize estradiol exhibit a plasma gonadotropin profile that is comparable to that of ovariectomized mice. However, we observed a number of striking differences in HPG axis function between ovariectomized wild-type females and those lacking ER
, as the latter exhibited the following: 1) normal Fshb expression and FSH secretion; 2) increased Gnrhr expression; 3) a greater increase in Cga expression; and 4) a lesser increase in Lhb expression and LH secretion.
The preservation of normal FSH regulation in ERKO and
ßERKO females, in contrast to the dramatic increases that follow ovariectomy in wild-type females, is perhaps easily explained. FSH synthesis and secretion are primarily regulated by the inhibin/activin family of hormones and is less dependent on estradiol feedback (21). The dimeric inhibin/activin hormones are composed from a pool of three individual subunits, inhibin-
(Inha), inhibin-ßA (Inhba), and inhibin-ßB (Inhbb), such that the possible combinations result in two potential forms of inhibin (
-ßA,
-ßB) and three potential forms of activin (ßA-ßA, ßB-ßB, ßA-ßB) (21). Herein, we found Inha to be highly expressed in the ovary relative to Inhba and Inhbb whereas the opposite was true in the pituitary (Fig. 6
). In the HPG axis, inhibin is the principal negative modulator of FSH synthesis and secretion, whereas activins promote the opposite. Therefore, increased FSH secretion after ovariectomy is likely the compound effect of the loss of ovarian-derived inhibin and hence negative modulation in the context of continued activin synthesis in the pituitary leading to positive modulation. In fact, we found that ovariectomized wild-type females exhibited a greater than 3-fold increase in pituitary Inhbb expression, an intriguing observation since activin-ßB has been reported to stimulate FSH secretion via a short loop within the pituitary (22). All three ERKO models exhibited normal Inha expression in the ovary or pituitary (Fig. 6A
), indicating that inhibin-mediated negative regulation of FSH secretion is preserved in the ERKO females and hence explaining the lack of increased FSH levels. Nonetheless, the question remains as to why mice lacking a functional Cyp19 gene exhibit a phenotype of FSH dysregulation similar to ovariectomy (20) whereas those lacking either ER form do not. There are currently no reports of inhibin/activin levels in the Cyp19 null mice.
Other disparities between ovariectomized wild-type females and those lacking ER may not be as easily rationalized, such as the opposite effect of each manipulation on Gnrhr expression in the pituitary. The level of Gnrhr expression in gonadotropes is highly dependent on the pulsatile secretion of GnRH from the hypothalamus, as constant perfusion with hormone leads to a degradation in receptor levels and eventual gonadotrope insensitivity (23, 24). Therefore, our finding that ovariectomized wild-type females exhibited reduced Gnrhr expression suggests that GnRH pulsatility is increased toward saturation after ovariectomy. In contrast,
ERKO and
ßERKO females exhibited significant increases in Gnrhr expression, indicating that GnRH pulsatility is not only preserved after ER
gene disruption but altered in a manner that promotes Gnrhr expression. It is currently believed that fluctuating GnRH receptor (GnRH-R) levels on the gonadotrope cell surface may be a mechanism by which the pituitary differentially responds to GnRH in terms of the quantity and nature of gonadotropin synthesis (25). Kaiser et al. (26) employed GH3 cells that express varied levels of GnRH-R to demonstrate that low receptor levels are conducive to induction of the Fshb promoter with little effect on the Cga and Lhb promoters; in contrast, increased GnRH-R levels favor Cga and Lhb expression while inhibiting Fshb. These data may explain the divergent phenotypes observed in ER
null mice vs. those lacking estradiol due to ovariectomy, as increased GnRH-R levels in the
ERKO and
ßERKO pituitary correlate with increased Cga and Lhb but normal Fshb expression, whereas ovariectomized wild-type females exhibited decreased Gnrhr but significantly increased Fshb expression.
The question remains as to the role of ERß in the female hypothalamus. Recent descriptions of estradiol binding and transcripts encoding ERß (5, 6) and estrogen-related receptor- (7) in GnRH-secreting neurons of the mouse hypothalamus challenge the long-held hypothesis that estrogen action in these cells is indirect. These reports, which also continue to claim that ER
remains undetectable in these same cells (7), provide the first experimental evidence that estrogen regulation of GnRH secretion may be direct and mediated by ERß. Although ERß expression is preserved in the
ERKO hypothalamus (27, 28), our findings provide little indication as to its possible function, as only females lacking ER
exhibited aberrant GnRH and LH secretion. However, the increased serum LH observed in
ßERKO females relative to
ERKO may indeed indicate an additive effect of the loss of both ERs in the hypothalamus as might be expected if the two act in parallel or in the form of a heterodimer (29, 30). This notwithstanding, our studies have focused on the role of the ERs in the maintenance of tonic gonadotropin levels in the female and provide little insight into the mechanism by which estradiol is involved in generating the LH surge that is paramount to ovulation and mating behavior. It is conceivable that ERß is directly involved in mediating such positive actions of estradiol in GnRH-secreting neurons, as ßERKO females exhibit reduced fecundity (13), a phenotype that could indeed be associated with abnormalities in frequency and amplitude of the LH surge.
Ovarian Steroidogenesis in the ERKO
Based on our knowledge of ER localization within the rodent ovary, a simplified model may state that any actions of estradiol in thecal cells are likely mediated by ER, whereas those in granulosa cells are likely mediated by ERß. For example, estradiol has been shown to inhibit LH-stimulated androgen synthesis in thecal cells (31, 32) while augmenting FSH-stimulated steroidogenesis in granulosa cells (2, 33); are these the actions of ER
and ERß, respectively? To address these questions, we evaluated the steroidogenic function of ovaries from all three lines of ERKO mice. This assessment of the gonadal portion of the HPG axis in ERKO females consisted of comparing the relative expression levels of the integral components of the steroidogenic pathway followed by measuring the pertinent steroid end products in the plasma.
A principal element of the two-cell, two-gonadotropin paradigm of ovarian steroidogenesis is that CYP17 is specific to the thecal cells, thereby forcing granulosa cells to be dependent upon the theca as the sole source of androgen precursors available for conversion to estradiol. Continuing with the simplified model of estrogen action in the ovary, our findings indicate no role for ER in the positive regulation of Cyp17 or any other gene products required for androstenedione synthesis in thecal cells. The same is true for ERß, as no aberrant phenotype in thecal cell enzymes or steroidogenesis was observed in ßERKO females. Rather, our data indicate that LH hyperstimulation of the ovary in
ERKO females leads to increased Cyp17 expression and androstenedione synthesis, as treatments with a GnRH antagonist were totally effective in relieving these phenotypes. This is not surprising, as positive regulation of Cyp17 expression in thecal cells is known to be primarily via LH, as effectively illustrated by the dramatic reduction in Cyp17 levels in the ovaries of LH-receptor null mice (34).
Several years ago, Leung and co-workers (31, 32) postulated that estradiol mediates an intraovarian feedback loop to maintain a proper balance of androgen synthesis. Therefore, it is possible that enhanced androgen synthesis in the ERKO ovary may also be due to a loss of estradiol-mediated suppression of Cyp17 expression or enzyme activity. Indeed, estrogens have been shown to inhibit CYP17 activity in Leydig cells while having no effect on the levels of upstream steroidogenic enzymes or LH receptor, but the mechanism remains elusive (3, 35, 36, 37). Early studies indicated estradiol competitively inhibits CYP17 enzymatic activity (37), whereas more recent studies report that cotreatment of rats with an antiestrogen blocked the effect of estradiol, suggesting an ER-dependent mechanism (38). Furthermore, estradiol has been shown to significantly reduce the level of Cyp17 transcripts in testes of rats (39) and fish (40), indicating an effect at the level of gene transcription. Our finding that
ERKO ovaries exhibit increased Cyp17 expression and enzyme activity, indicated by elevated plasma androstenedione, in a milieu of enriched estradiol levels, argues against direct inhibition of CYP17 activity but instead supports an ER
-mediated repression of Cyp17 gene transcription in thecal cells.
Normal Cyp19 and Hsd17b1 expression in ßERKO ovaries excludes any critical role of ERß in the positive regulation of estradiol synthesis in granulosa cells that may have been postulated by the above model. Furthermore, elevated Cyp19 and Hsd17b1 expression in ERKO ovaries suggests ER
may, in fact, negatively modulate estradiol synthesis, despite being the less expressed ER in granulosa cells. However, GnRH antagonist treatments completely abrogated the increased Cyp19 and Hsd17b1 expression characteristic of
ERKO ovaries, indicating these phenotypes are subordinate to the loss of ER
in the HP axis rather than the ovary. However, most evidence indicates that direct actions of LH on granulosa cells actually reduce aromatase activity (2, 33), making it unlikely that increased Cyp19 expression in
ERKO ovaries is a direct effect of elevated LH, therefore compelling the need to consider other possibilities. For example, estradiol is known to augment the stimulatory actions of FSH on Cyp19 expression in granulosa cells but has little effect when acting alone (2, 33). Therefore, it is conceivable that heightened estradiol levels in the
ERKO are indeed acting via ERß to enhance FSH induction of Cyp19, a scenario that is supported by our previous reports of normal ERß and elevated FSH receptor expression in
ERKO ovaries (41). The return of Cyp19 expression to normal after the loss of both ERs in the
ßERKO ovary further supports this possibility. It is also plausible that the elevated androgens in the
ERKO due to LH hyperstimulation of the theca contribute to increased granulosa cell steroidogenic enzyme expression, as both testosterone and dihydrotestosterone are reported to enhance FSH induction of Cyp19 (33, 42, 43) and Hsd17b1 (44, 45). Interestingly, FSH receptor-null mice maintain normal Cyp19 expression in the context of elevated testosterone (46), suggesting that androgens can indeed preserve Cyp19 expression in the absence of FSH action. Herein, flutamide treatment of
ERKO females did slightly reduce Cyp19 expression but was not nearly as effective as the GnRH antagonist. Lastly, increased steroidogenic enzyme expression in
ERKO granulosa cells may also be the result of LH-stimulated overproduction of a paracrine-acting factor from the theca. Regardless, it must be recognized that all three above scenarios involve a gonadotropin component and therefore would be attenuated by GnRH antagonist treatment.
Interestingly, ovaries from ßERKO females did not exhibit increased Cyp17 or Cyp19 expression, or the respective steroid end products, despite exhibiting a gonadotropin profile comparable to the
ERKO. Although these data suggest the two ERs may cooperate within the ovary to allow for proper gonadotropin regulation of steroidogenic enzyme expression, this finding must be considered in the context of the unique ovarian phenotype of
ßERKO females (47). Adult
ßERKO ovaries exhibit massive oocyte death, a reduced granulosa cell population, and the overt presence of Sertoli-like cells (47, 48). Therefore, it is equally plausible that the absence of an
ERKO-like increase in Cyp19 expression in the
ßERKO ovaries may be representative of granulosa cell attrition rather than an inability to properly respond to gonadotropins. Because no frank thecal cell phenotype has been described in the
ßERKO ovary, it is also surprising that no
ERKO-like increase in Cyp17 expression was observed, despite these females possessing plasma LH levels that were more than 2-fold higher than
ERKO levels. It is plausible that the dramatic loss of granulosa and germ cells may also impact LH responsiveness in thecal cells as multiple granulosa and germ cell-derived paracrine-acting factors, such as IGF-1 and growth differentiation factor-9, respectively (49, 50), are known to positively influence thecal cell functions. These and other such factors would presumably be reduced in
ßERKO ovaries.
The most novel finding was the discovery of Hsd17b3 gene expression in the ovaries of ERKO and
ßERKO females. The encoded enzyme, 17ßHSD type III, specifically mediates the reduction of androstenedione to testosterone (11) and is reported to be unique to the Leydig cells of the testes (51, 52, 53, 54), as we also found to be true when comparing wild-type gonads of both sexes (Fig. 3
). Therefore, our finding of Hsd17b3 expression in
ERKO and
ßERKO ovaries at levels comparable to those in wild-type testes was quite surprising. Foremost, these data indicate that elevated plasma testosterone levels in
ERKO and
ßERKO females are due to an active synthesis within the ovaries and suggest that ER
may be involved in repressing Hsd17b3 expression in the wild-type ovary. These findings also extend previous descriptions of the
ßERKO ovarian phenotype (47, 48) to now include a phenotype of endocrine sex reversal as well as represent the first report of a similar phenotype in the
ERKO ovary. Since assays to localize the Hsd17b3 expression in the
ERKO and
ßERKO ovaries were not carried out, we can only speculate that expression may be occurring in thecal cells, as they are the ovarian counterpart to Leydig cells. However, we cannot exclude the ectopic presence of Leydig-like cells in the
ERKO and
ßERKO ovaries, as such a phenotype was recently described in the ovaries of Cyp19 null mice (55). Interestingly, there exists a single report of HSD17b3 expression in a human Sertoli-Leydig cell tumor isolated from a 46,XY female (56).
Although little is known about the regulation of Hsd17b3 expression in Leydig cells, Baker et al. (52) demonstrated that the expected postpubertal rise in expression does not occur in the testes of hypogonadal (hpg) and testicular-feminized (tfm) mice, indicating that positive regulation of Hsd17b3 expression is dependent upon gonadotropin and/or androgen stimulation. Our data indicate that chronic hyperstimulation of ERKO and
ßERKO ovaries by LH is a major factor in the ectopic expression of Hsd17b3 as GnRH-antagonist treatments completely abolished detectable expression. However, our preliminary evaluation of ovaries from transgenic mice that possess elevated LH levels (57) indicates no Hsd17b3 expression (Couse, J. F., J. H. Nilson, and K. S. Korach, manuscript in preparation), suggesting that chronic LH stimulation of the wild-type ovary is not sufficient to induce Hsd17b3 expression but only occurs in the context of an ovary that is lacking functional ER
. The influence of increased androgens on Hsd17b3 expression was also apparent as flutamide treatment of
ERKO females significantly reduced expression, although this treatment proved not as effective as the GnRH antagonist.
Lastly, there are multiple similarities between the ERKO ovarian phenotype and the clinical features of human polycystic ovarian syndrome (PCOS), or functional ovarian hyperandrogenism (FOH), as this syndrome is more precisely defined (58). FOH is characterized as gonadotropin-dependent dysregulation of androgen secretion from the ovary (58). The etiology of FOH is speculated as being due to either excessive LH stimulation, an escape from LH-induced desensitization of the ovarian theca after ovulation, or a steroidogenic block within the ovary that allows for the accumulation of androgen precursors (58). We propose that the
ERKO may represent the former two causal factors, as these mice are anovulatory, lack corpora lutea, and exhibit chronically elevated plasma LH and increased LH receptor in the ovary (14, 41). Mice lacking Cyp19 also exhibit an
ERKO-like ovarian morphology of multiple cystic follicles as well as elevated plasma gonadotropins and androgens (20, 55) and may perhaps be considered analogous to the later cause of FOH. Herein, we show that
ERKO ovaries also exhibit a generalized increase in steroidogenic capacity, most notably CYP17 and CYP19 that is indeed gonadotropin dependent. Recent in vitro studies of thecal cells from PCOS patients have demonstrated increased basal and cAMP-induced expression of CYP17 relative to normal thecal cells (59, 60). Furthermore, Nelson et al. (59) described the metabolic profile of PCOS thecal cells in culture to be indicative of the presence of HSD17b3; however, later studies found no detectable expression (61). Previously reported nonreproductive similarities observed in the
ERKO female include obesity and insulin resistance (62), both of which are common in human PCOS (63). In fairness, one major difference is that
ERKO females do not exhibit the thickened outer capsule that is characteristic of human PCOS ovaries (63).
In summary, our findings indicate that ER is indispensable in mediating the negative-feedback effects of estradiol necessary to maintain homeostasis of LH synthesis and secretion from the pituitary in the female mouse, whereas the loss of ERß appears to have minimal impact on basal gonadotropin gene expression and secretion. This increase in LH in the
ERKO females is the primary causal factor of increased levels of Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary and therefore the abnormal steroid hormone milieu characteristic of these animals. These studies extend our previous morphological descriptions of the
ERKO ovary and provide further evidence that the most dramatic ovarian phenotypes in the
ERKO are due to chronic LH stimulation and not inherent to the loss of intraovarian ER
function. Interestingly, the manifestations of LH hyperstimulation in the
ERKO ovary may, in fact, depend on the presence of functional ERß as
ßERKO females exhibit a similar gonadotropin profile but lack consistent evidence of enhanced estradiol synthesis. The discovery of ectopic Hsd17b3 expression in
ERKO and
ßERKO ovaries extends the sex reversal phenotype in the
ßERKO ovary to now include an endocrine phenotype and marks the first such description of a similar phenotype in
ERKO ovaries.
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MATERIALS AND METHODS |
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All mice were 24 months of age with the exception of the ßERKO mice used for gene expression assays, which ranged from 39 months of age. Where applicable, certain experiments included ovariectomized wild-type mice as a sex steroid-deficient model, in which case animals were ovariectomized 1214 d before sample collection. In those experiments utilizing a GnRH antagonist, animals were treated sc with 60 µg Antide (Sigma, St. Louis, MO) or vehicle (20% propylene in 0.85% saline) in 0.1 ml volume at 12001300 h every 48 h for a total of six treatments as previously described (14); tissues were collected 24 h after the final treatment. In those experiments utilizing the androgen antagonist, flutamide, animals were implanted sc with a single 21-d release pellet of 50 mg flutamide (Innovative Research of America, Sarasota, FL) or placebo, and tissues were collected 15 d later. Those animals receiving both flutamide and GnRH-antagonist treatments were first implanted with the flutamide pellet followed by Antide injections beginning 3 d later. All mice were euthanized by carbon dioxide asphyxiation, and whole blood was collected from the inferior vena cava and heparinized, and the plasma was stored at -70 C until assayed. Samples to be assayed for PRL content were specifically collected from animals that were single housed and euthanized by decapitation within 30 sec of removal from the cage to minimize the effects of acute stress on the animal (16). In all cases, pituitary and ovary (trimmed of oviduct and surrounding tissue) were immediately collected, weighed, and frozen at -70 C in preparation for RNA extraction.
Total RNA Isolation
Total RNA was isolated from frozen tissues using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturers protocol. The concentration of all final preparations was calculated via an A260 reading using a DU-640 UV spectrophotometer (Beckman Coulter, Inc., Fullerton, CA) and a 0.5- to 1-mg aliquot was analyzed on a 1% agarose gel to ensure RNA integrity before further analysis.
Gene Expression Assays
Northern Blot and RPA.
All gene expression assays were carried out on RNA preparations from individual animals. Quantitative analysis of highly expressed genes was achieved via Northern blot analysis (see Table 1). In each case, a Northern blot of 2 µg total RNA per sample was generated using the NorthernMax formaldehyde-based reagents and BrightStar (Ambion, Inc., Austin, TX), positively charged nylon membrane according to the manufacturers protocol. All blots were first probed for the experimental gene of interest, stripped according to the manufacturers protocol, and then reprobed for normalization. Quantitative expression assays of several other transcripts were carried out by RPA using the Hybspeed RPA kit (Ambion, Inc.) according to the manufacturers protocol. The following probe sets were designed to allow for a single-tube RPA (see Table 1
for further information): 1) Lhb, Fshb with Rps28 for normalization; 2) Cyp11a, Hsd3b1, Cyp17, Cyp19 with Ppia (cyclophilin A) for normalization; 3) Hsd17b1, Hsd17b3 with Actb for normalization; 4) Inha, Inhba, Inhbb, Fst with Ppia for normalization. All other genes assayed by RPA but not listed above were carried out separately (see Table 1
). The amount of total RNA used per assay ranged from 520 µg depending on the probe set and experiment. Hybridization times ranged from 11.5 h. All RPA samples were electrophoresed on precast 6% bis-acrylamide/8 M urea/1x Tris-borate-EDTA gels (Invitrogen), which were then fixed and dried in an Easy Breeze Gel Dryer (Amersham Pharmacia Biotech, Piscataway, NJ). Final Northern blots and RPA gels were exposed to a PhosphorImager screen and the data were analyzed with a Storm 860 and accompanying ImageQuant Software (Molecular Dynamics, Inc., Sunnyvale, CA), followed by exposure to x-ray film.
All Northern blots and RPAs employed radiolabeled antisense riboprobes generated from cDNA clones using Maxiscript reagents (Ambion, Inc.) and the appropriate [32P-]nucleoside triphosphate (Amersham Pharmacia Biotech). Probes intended for use on Northern blots were labeled using the Strip-EZ (Ambion, Inc.) system to allow for later stripping and reprobing with a probe specific to the selected normalization gene. Several probes that were not available otherwise were generated by cloning a PCR-amplified cDNA fragment of the respective mouse gene into the pCRII-TOPO vector (Invitrogen) according to the manufacturers protocol. All clones not previously described were confirmed by either sequencing or Northern blot analysis of RNA from known positive tissues or both.
SQ-RT-PCR.
As mentioned, all gene expression assays were carried out on RNA preparations from individual animals; however, for certain genes the level of expression or the amount of available RNA was limiting. In these cases, we used an SQ-RT-PCR approach to assess the level of gene expression (see Table 1). All RNA samples intended for assay by SQ-RT-PCR were rid of contaminating DNA using the DNA-free reagents (Ambion, Inc.) according to the manufacturers protocol, followed by an A260 reading using a Beckman Coulter, Inc. DU-640 UV spectrophotometer and final normalization to a concentration of 0.2 µg/ml. For each sample, 1 µg of RNA was used in a 25 µl cDNA reaction using random hexamers and the Superscript cDNA synthesis system (Invitrogen) according to the manufacturers protocol. PCR reactions were prepared using 1 µl cDNA per reaction for each respective primer set (Table 1
) in a 15-µl total reaction volume using PCR reagents and Platinum Taq Polymerase from Invitrogen. PCR was carried out in a Thermo Hybaid Multiblock System (Franklin, MA) with the following cycling conditions: 95 C/30 sec, 58 C/45 sec, 72 C/30 sec. Primers for ribosomal protein L7 (Rpl7) (Table 1
) were included in all reactions as a positive control and for normalization. Duplicate sets of samples were prepared and one set was run for 26 PCR cycles and the second was run for 32 PCR cycles. All samples were then electrophoresed on an agarose gel (2% NuSieve/0.7% SeaKem, BMA Bioproducts, Rockland, ME) in 1x Tris-borate-EDTA and then electoblotted to BrightStar nylon membrane (Ambion, Inc.) using the Royal Genie Blotter (Idea Scientific Co., Minneapolis, MN). All blots were then probed with an oligo specific to sequences nested within the PCR primers that was 5'-radiolabeled with [33P-
ATP] using T4 polynucleotide kinase (Amersham Pharmacia Biotech). Hybridization was carried out in Rapid-Hyb buffer (Amersham Pharmacia Biotech) with more than 3 x 106 cpm of probe/ml overnight, followed by washing according to the manufacturers protocol. Final SQ-RT-PCR blots were exposed to a PhosphorImager screen and the data analyzed with a Storm 860 and accompanying ImageQuant Software (Molecular Dynamics, Inc.).
Hormone RIAs
All hormone assays were carried out on plasma collected from individual animals. Plasma was processed from whole blood collected from the descending aorta (gonadotropin and steroid assays) or decapitation (PRL assays) upon euthanization and stored at -70 C until analysis. Plasma LH, FSH, and PRL levels were assessed by RIA in singlicate at 100 µl plasma per animal using the Biotrak 125I-RIA kits (Amersham Pharmacia Biotech). Plasma androstenedione, estradiol, and testosterone were assayed in singlicate per animal on 50-µl, 200-µl, and 50-µl aliquots, respectively, using the Active Androstenedione RIA, Ultra-Sensitive Estradiol Double-Antibody RIA, and Active Testosterone RIA kits (Diagnostics Systems Laboratories, Inc., Webster, TX) according to the manufacturers protocol. All final assay samples were quantified using a Packard Multi-Prias 2 counter (Packard Instruments, Downers Grove, CT). To avoid interassay variation, all assays were carried out in a single set-up with the exception of those for LH. The following parameters apply to the above stated RIAs (least detectable concentration; intrassay variability; interassay variability): LH (0.8 ng/ml; 7.4%; 10.4%); FSH [0.8 ng/ml; 5.8%; not applicable (n.a.)]; PRL (0.8 ng/ml; 11.5%, n.a.); androstenedione (0.8 ng/ml; 18.2%; n.a.), estradiol (2.2 pg/ml; 8.0%; n.a.); and testosterone (0.8 ng/ml; 9.3%; n.a.).
Statistical Analysis
All data sets were first tested for homoscedasticity of variance using Levenes test. In cases where data sets failed the Levenes test, data were log transformed before further statistical analysis. All data were assessed for statistically significant differences via a one-way ANOVA followed by the Fishers protected least-significant differences post hoc test. All statistical analyses were carried out using Statview 4.0 Software (SAS Institute, Inc., Cary, NC).
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FOOTNOTES |
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Received for publication December 2, 2002. Accepted for publication February 26, 2003.
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