Characterization of the Hypothalamic-Pituitary-Gonadal Axis in Estrogen Receptor (ER) Null Mice Reveals Hypergonadism and Endocrine Sex Reversal in Females Lacking ER{alpha} But Not ERß

John F. Couse, Mariana M. Yates, Vickie R. Walker and Kenneth S. Korach

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To determine the role of each estrogen receptor (ER) form (ER{alpha}, ERß) in mediating the estrogen actions necessary to maintain proper function of the hypothalamic-pituitary-gonadal axis, we have characterized the hypothalamic-pituitary-gonadal axis in female ER knockout (ERKO) mice. Evaluation of pituitary function included gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl. Evaluation of ovarian steroidogenic capacity included gene expression assays for the components necessary for estradiol synthesis: i.e. Star, Cyp11a, Cyp17, Cyp19, Hsd3b1, and Hsd17b1. These data were corroborated by assessing plasma levels of the respective peptide and steroid hormones. {alpha}ERKO and {alpha}ßERKO females exhibited increased pituitary Cga and Lhb expression and increased plasma LH levels, whereas both were normal in ßERKO. Pituitary Fshb expression and plasma FSH were normal in all three ERKOs. In the ovary, all three ERKOs exhibited normal expression of Star, Cyp11a, and Hsd3b1. In contrast, Cyp17 and Cyp19 expression were elevated in {alpha}ERKO but normal in ßERKO and {alpha}ßERKO. Plasma steroid levels in each ERKO mirrored the steroidogenic enzyme expression, with only the {alpha}ERKO exhibiting elevated androstenedione and estradiol. Elevated plasma testosterone in {alpha}ERKO and {alpha}ßERKO females was attributable to aberrant expression of Hsd17b3 in the ovary, representing a form of endocrine sex reversal, as this enzyme is unique to the testes. Enhanced steroidogenic capacity in {alpha}ERKO ovaries was erased by treatment with a GnRH antagonist, indicating these phenotypes to be the indirect result of excess LH stimulation that follows the loss of ER{alpha} in the hypothalamic-pituitary axis. Overall, these findings indicate that ER{alpha}, but not ERß, is indispensable to the negative-feedback effects of estradiol that maintain proper LH secretion from the pituitary. The subsequent hypergonadism is illustrated as increased Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ENDOCRINE FEEDBACK loops that provide for integrated function among the organs of the hypothalamic-pituitary-gonadal (HPG) axis are paramount to reproductive potential (1). GnRH from the hypothalamus stimulates the anterior pituitary to secrete FSH and LH, which act on the ovary to promote folliculogenesis and the concomitant synthesis of estradiol. In turn, estradiol is considered the critical determinant of plasma gonadotropin levels in the female by completing an endocrine feedback loop upon the hypothalamus to reduce GnRH secretion. Estrogen is also reported to act in an auto- or paracrine manner to influence certain ovarian functions, most notable of which are an enhancement of FSH action on granulosa cells (2), inhibition of androgen synthesis in thecal cells (3), and an attenuation of apoptosis (4).

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{alpha} 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{alpha} 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 ({alpha}ERKO), or Esr2 gene (ßERKO), or both ({alpha}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 {alpha}-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)/{Delta}5-{Delta}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-{alpha} (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{alpha} but not ERß is essential in mediating the negative-feedback effects of estradiol on the hypothalamic-pituitary (HP) axis, as only {alpha}ERKO and {alpha}ßERKO females exhibited elevated LH synthesis and secretion. The resulting hyperstimulation of the ovary in {alpha}ERKO females leads to increased Cyp17, Cyp19, and Hsd17b1 expression, which ultimately manifests as elevated levels of plasma androstenedione and estradiol. However, {alpha}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 {alpha}ERKO. Most interesting was an apparent endocrine sex reversal observed in {alpha}ERKO/{alpha}ßERKO ovaries, characterized by the ectopic expression of Hsd17b3, an enzyme-encoding gene known to be specifically expressed in testes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadotropin and PRL Regulation in ERKO Female Pituitary
Evaluation of anterior pituitary function in ERKO females consisted of gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl (Table 1Go); followed by assays for the plasma levels of the respective hormones. Ovariectomized wild-type females were included in all assays as a sex steroid-deficient model. The average plasma gonadotropin and PRL levels are shown in Table 2Go. As expected, ovariectomy in wild-type females led to a significant increase in plasma LH and FSH, of 4- and 7-fold wild type, respectively. Comparable increases in circulating LH were observed in intact {alpha}ERKO (3-fold) and {alpha}ßERKO females (6-fold), whereas ßERKO females exhibited wild type-like levels. Interestingly, plasma LH levels in the {alpha}ERKO were significantly lower than that exhibited by ovariectomized wild-type females (P < 0.024), suggesting that preservation of ERß functions in the HP axis may provide some degree of negative feedback in the {alpha}ERKO. This is supported by the increased plasma LH exhibited in the {alpha}ßERKO females, which was more than 2-fold that of the {alpha}ERKO and more comparable to the wild-type ovariectomized group. In contrast to the effects of a lack of ER{alpha} on LH regulation, plasma FSH levels remained normal in all three ERKO lines, whereas ovariectomized wild-type females exhibited a more than 6-fold increase.


View this table:
[in this window]
[in a new window]
 
Table 1. Specifications of All Probes and PCR Primers Used

 

View this table:
[in this window]
[in a new window]
 
Table 2. Plasma Hormone Levels in Untreated Adult Females or in Those Following Treatment with a GnRH Antagonist

 
The plasma hormone levels described above were mirrored in the assays for gonadotropin subunit gene expression in the anterior pituitary. As shown in Fig. 1Go, Lhb expression in {alpha}ERKO and {alpha}ßERKO females was elevated relative to wild type (P < 0.0001) and more comparable to that observed in ovariectomized wild-type females. Similar increases in Cga expression were exhibited by the {alpha}ERKO and {alpha}ßERKO but were not observed in ovariectomized wild-type females. In contrast, ßERKO females exhibited normal regulation of both Cga and Lhb in the pituitary. Congruent with the normal plasma FSH levels found in each ERKO line, Fshb levels in the pituitary of all three ERKOs were within wild-type limits whereas the wild-type ovariectomized group exhibited a dramatic increase. Overall, these data indicate that estradiol-mediated negative regulation of Cga and Lhb gene transcription as well as LH secretion are heavily dependent upon the actions of ER{alpha}, whereas Fshb expression and FSH secretion were not affected by the loss of either ER form.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 1. Gonadotropin-Related Gene Expression in the Pituitary of ERKO Females

Shown are representative gene expression assays for Gnrhr, Cga, Lhb, and Fshb in pituitary of individual wild-type (WT-int) and ERKO females. Ovariectomized wild-type (WT-ovx) females were included as sex steroid-deficient models. Below are bar graphs illustrating the average (±SEM) gene expression for each gene and genotype, expressed as fold wild-type intact after normalization to Rps28 levels. All data were generated from samples collected from individual animals; n = 6–12 for all genotypes and assays (except Cga in {alpha}ßERKO = 5). a, P < 0.05 vs. WT-int; b, P < 0.05 vs. WT-ovx; c, P < 0.05 within ERKO genotypes.

 
Evaluation of Gnrhr expression in the anterior pituitary indicated statistically significant increases in the {alpha}ERKO (P < 0.0001) and {alpha}ßERKO (P < 0.0178) females, whereas expression in the ßERKO females was comparable to wild type (Fig. 1Go). Interestingly, ovariectomy in wild-type females had the opposite effect on Gnrhr expression, eliciting a statistically significant decrease compared with intact wild type (P < 0.0266).

The effect of ovariectomy and ER gene disruption on Prl expression in the anterior pituitary is shown in Fig. 2Go. 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 {alpha}ERKO and {alpha}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{alpha} was not reflected in plasma PRL levels, which were slightly reduced in each but within normal range (Table 2Go). 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 {alpha}ßERKO plasma.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 2. Prl Gene Expression in the Pituitary of ERKO Females

Shown is a representative Northern blot for Prl in pituitary of individual wild-type (WT-int) and ERKO females. Ovariectomized wild-type (WT-ovx) females were included as sex steroid-deficient models. Below is a bar graph illustrating the average (±SEM) for Prl gene expression in each genotype, expressed as fold wild-type intact after normalization to Rps28 levels. All data were generated from samples collected from individual animals; n = 6–12 for all genotypes except {alpha}ßERKO = 5. a, P < 0.05 vs. WT-int; b, P < 0.05 vs. WT-ovx; c, P < 0.05 within ERKO genotypes.

 
Steroidogenesis in ERKO Ovaries
Figure 3Go illustrates the two-cell, two-gonadotropin paradigm of steroidogenesis in the mammalian ovary, as described by Gore-Langton and Armstrong (10). For the purpose of clarity, only the {Delta}5 pathway of androgen synthesis in thecal cells is shown. The {Delta}4 steroidogenic pathway (not shown) involves the same enzymes and terminates with androstenedione as the final product but follows a different sequence of enzyme action. Our evaluation of steroidogenesis in ERKO ovaries consisted of gene expression assays for the steroidogenic enzymes necessary for de novo synthesis of estradiol from cholesterol: Cyp11a (CYP11A), Cyp17 (CYP17), Hsd3b1 (3ßHSD type I), Cyp19 (CYP19), and Hsd17b1 (17ßHSD type I) (Table 1Go). Assays for expression of the Star (StAR) gene were included because of its critical role in cholesterol mobilization to the inner mitochondrial membrane, the site of CYP11A activity. In addition, we assessed plasma levels of the pertinent steroid end-products of each follicular unit, these being androstenedione from the theca and estradiol from the granulosa. Also shown in Fig. 3Go is the enzymatic action of the Hsd17b3 gene product (17ßHSD type III), which specifically converts androstenedione to testosterone. This enzyme is not considered a component of the two-cell, two-gonadotropin model of ovarian steroidogenesis (11) but is shown for reasons to be clarified later.



View larger version (55K):
[in this window]
[in a new window]
 
Figure 3. Gene Expression Assays for Steroidogenic Components in the ERKO Ovary

Top left, The enzymatic pathway of the two-cell, two gonadotropin paradigm of steroidogenesis in the ovary is shown in blue (10 ). The enzymatic action of 17ßHSD III is shown in red to indicate that this enzyme is not a component of the two-cell, two-gonadotropin paradigm as it is unique to the testis. Top right, Representative RPAs for transcripts encoding Star and the steroidogenic enzymes in wild-type (WT) and ERKO ovaries. Testes (T) were included in assays for Hsd17b1 and Hsd17b3 as a negative and positive control, respectively. The arrow to the right of the Cyp19 panel is intended to designate the protected fragment representative of Cyp19; the slightly larger band is a background anomaly derived from the Cyp17 probe. Bottom, Bar graphs illustrating the average (±SEM) gene expression for each gene and genotype, expressed as fold wild-type intact after normalization to Ppia (cyclophilin A) or Actb levels; the exception is Hsd17b3, which is shown as % Actb because this mRNA is not detected in wild-type ovary. All data were generated from samples collected from individual animals; n >= 6 for all genotypes in each assay except the wild-type testis, n = 3. a, P < 0.05 vs. WT-int; b, P < 0.05 vs. WT-ovx; c, P < 0.05 within ERKO genotypes.

 
The results of the gene expression assays are summarized in Fig. 3Go. ERKO ovaries from all three lines exhibited no dysregulation of the Star gene. Furthermore, ßERKO and {alpha}ßERKO females exhibited wild-type levels of the steroidogenic enzymes necessary for the conversion of cholesterol to estrone, a weak estrogen product. In contrast, {alpha}ERKO ovaries invariantly exhibited an almost 3-fold increase in the expression of Cyp17 and Cyp19 compared with wild type (P < 0.0001), whereas Cyp11A and Hsd3b1 levels remained relatively normal. Ovaries from {alpha}ßERKO females also exhibited a trend toward increased Cyp19 expression (Fig. 3Go), but a high degree of variability within this group negated any statistical significance. All three ERKO lines exhibited increased expression of Hsd17b1. Plasma levels of sex steroids in each ERKO female (Table 2Go) strongly agree with the steroidogenic enzyme profile indicated in the gene expression assays. The average plasma androstenedione and estradiol levels in {alpha}ERKO females were 3- and 8-fold that of age-matched wild-type females, respectively. In contrast, plasma androstenedione and estradiol levels in the ßERKO and {alpha}ßERKO were within the wild-type range. This strong correlation between steroidogenic enzyme expression and plasma steroid levels is congruent with previous reports that the level of enzyme expressed is highly predictive of the steroid ultimately produced (12).

As previously reported (13) and repeated here, {alpha}ERKO females exhibited elevated plasma testosterone levels relative to wild-type (30-fold), which approach the minimum level of detection (Table 2Go). A more modest but significant (15-fold) increase in plasma testosterone was exhibited by {alpha}ßERKO females. However, our evaluation of steroidogenic enzyme expression in {alpha}ERKO and {alpha}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. 3Go). We therefore assayed for expression of the Hsd17b3 gene, which encodes the enzyme that preferentially converts androstenedione to testosterone (see Fig. 3Go). 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 {alpha}ERKO and {alpha}ßERKO ovaries, but absent in the ßERKO. In fact, the levels of Hsd17b3 expression in the {alpha}ERKO and {alpha}ßERKO ovaries were comparable to that found in adult wild-type testis (Fig. 3Go).

Effect of a GnRH Antagonist and Antiandrogen on the {alpha}ERKO HPG Axis
We have previously shown that treatment of {alpha}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 {alpha}ERKO ovary (14). To more fully characterize the effects of such treatment in the {alpha}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 {alpha}ERKO and ovariectomized wild-type females (Fig. 4Go). Similar treatments were also effective in reducing the elevated Fshb expression that occurs after ovariectomy in wild-type females (Fig. 4Go). Accordingly, plasma LH and FSH levels in GnRH antagonist-treated {alpha}ERKO and ovariectomized wild-type females were reduced to within the range of untreated intact wild-type females (Table 2Go), 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{alpha}.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 4. Effect of a GnRH Antagonist on Pituitary and Ovarian Gene Expression in Wild-Type and {alpha}ERKO Females

A (left), Shown are representative gene expression assays for Cga, Fshb, and LHb in pituitary of intact wild-type (WT-int) and {alpha}ERKO females treated with vehicle (V) or GnRH-antagonist (A). Ovariectomized wild-type (WT-ovex) females were included as a sex steroid-deficient model. A (right), Bar graphs summarizing gene expression data for each respective gene assayed in the pituitary; illustrated as fold wild type intact, vehicle treated. B (left), Shown are representative gene expression assays for Cyp17, Cyp19, Hsd17b1, and Hsd17b3 in ovaries of wild-type (WT) and {alpha}ERKO females treated with vehicle or the GnRH antagonist. B (right), Bar graphs illustrating the average (±SEM) gene expression for each gene, genotype, and treatment, expressed as fold wild type intact after normalization to Rps28 or as % Actb levels. All data were generated from samples collected from individual animals; n >= 4 for all groups except the {alpha}ERKO (V) pituitary, n = 2. a, P < 0.05 vs. WT-int or WT-vehicle; b, P < 0.05 when comparing treatment within genotype and gonadal status.

 
To determine the extent to which the steroidogenic phenotypes exhibited by the {alpha}ERKO ovaries are the secondary effects of excess LH, we evaluated the effect of GnRH antagonist treatment on steroidogenic enzyme expression in {alpha}ERKO ovaries. As shown in Fig. 4BGo, GnRH antagonist-treated {alpha}ERKO females exhibited reductions in Cyp17, Cyp19, and Hsd17b1 expression to levels that were comparable to untreated wild-type females. Furthermore, GnRH antagonist treatment completely abolished the ectopic expression of Hsd17b3 in {alpha}ERKO ovaries (Fig. 4BGo). Because a primary action of LH in thecal cells is to stimulate androgen production (3), and androgens reportedly augment their own production by enhancing Cyp17 and Hsd17b3 expression in testis, it is conceivable that the increased Cyp17 and ectopic Hsd17b3 expression in {alpha}ERKO ovaries is secondary to LH hyperstimulation and more directly attributable to subsequent increases in local androgens. To assess this possibility, we evaluated the expression of Cyp17, Cyp19, Hsd17b1, and Hsd17b3 in wild-type and {alpha}ERKO ovaries after 2 wk of treatment with the antiandrogen, flutamide. Similarly treated wild-type males were included as a positive control, and seminal vesicles, a highly androgen-dependent tissue, were weighed to assess the effectiveness of the treatment. As expected, 2 wk of flutamide exposure significantly reduced seminal vesicle weight (P < 0.038) in wild-type males (0.76% body weight) compared with placebo treated wild-type males (1.02% body weight). Interestingly, GnRH antagonist treatments proved more effective in reducing seminal vesicle weight (0.40% body weight; P < 0.0004), likely due to decreasing LH-induced androgen synthesis as no additive effect of flutamide plus GnRH antagonist was observed (0.43% body weight). In {alpha}ERKO ovaries, flutamide and GnRH antagonist were equally effective in reducing Cyp17 expression in the {alpha}ERKO ovary to within the wild-type range (Fig. 5Go). Flutamide treatment also considerably decreased Hsd17b3 expression in the {alpha}ERKO (P < 0.038 vs. untreated {alpha}ERKO) but was not as effective as the GnRH antagonist, which completely abolished all detectable expression. In contrast to its effects on thecal cell enzymes, flutamide treatment had minimal impact on Cyp19 expression and, in fact, caused an increase in Hsd17b1 expression in wild-type ovaries. All effects on steroidogenic enzyme expression after combined flutamide and GnRH antagonist treatment were similar to those of the GnRH antagonist alone (data not shown). Evaluation of the effect of GnRH antagonist and flutamide treatment on the expression of Hsd17b3 in {alpha}ßERKO ovaries was not possible due to limited animal availability.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 5. Effect of Flutamide on Ovarian Gene Expression in {alpha}ERKO Females

Bar graphs illustrating the average (±SEM) gene expression in the ovary after treatment with either vehicle, GnRH-antagonist (Antide), or flutamide, expressed as % Actb levels. All data were generated from samples collected from individual animals; n >= 4 for all groups. a, P < 0.05 vs. WT-vehicle; b, P < 0.05 when comparing treatments within genotype. nd, None detected.

 
Expression of Inhibin Subunits and Follistatin in ERKO Pituitary and Ovary
To better understand the normal FSH levels in the ERKO females, we sought to characterize the expression levels of the inhibin subunit and follistatin-encoding genes in the ovary and pituitary. In general, we found transcripts encoding the inhibin subunits (Inha, Inhba, Inhbb) and Fst to be easily detectable in the ovary of all genotypes, with Inha exhibiting the highest level relative to the others (Fig. 6Go). Relatively normal expression of all four genes was observed in ERKO ovaries, with the exception of a significant increase in Inhbb expression in the ßERKO and {alpha}ßERKO. In the pituitary, Fst transcripts were undetectable by semiquantitative RT-PCR (SQ-RT-PCR) and Inha expression was much lower relative to the ovary and exhibited no effect of ovariectomy or ER gene disruption. In contrast, transcripts encoding the subunits, Inhba and Inhbb, were easily detectable by SQ-RT-PCR in the pituitaries of each group and showed drastically different effects of ovariectomy vs. the loss of ER{alpha} function. Pituitary Inhba expression was significantly elevated 2.5-fold (P < 0.0001 vs. wild type) in {alpha}ERKO and {alpha}ßERKO females but normal in ovariectomized wild types (Fig. 6Go). In contrast, pituitary Inhbb expression was significantly up-regulated after ovariectomy in wild-type females (P < 0.0001 vs. wild-type), but only slightly increased in the {alpha}ERKO and {alpha}ßERKO pituitaries, while remaining normal in the ßERKO.



View larger version (32K):
[in this window]
[in a new window]
 
Figure 6. Evaluation of Inha, Inhba, Inhbb, and Fst Expression in Pituitary and Ovary of ERKO Females

Shown are representative gene expression assays for Inha, Inhba, Inhbb, and Fst in ovary (A) and pituitary (B) as detected by RPA and SQ-RT-PCR, respectively. All assays were carried out on individual intact wild-type (WT-int), wild-type ovariectomized (WT-ovx), and ERKO females. Right, Table showing the average (±SEM) gene expression for each gene and genotype, expressed as fold wild type after normalization to Actb or RpL7 levels. All data were generated from samples collected from individual animals; n = 6 for all groups. n.a., Not applicable. a, P < 0.05 vs. WT or WT-int; b, P < 0.05 vs. WT-ovx; c, P < 0.05 among ERKOs.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although both ER{alpha} and ERß are present throughout the organs of the HPG axis, they exhibit a distinct expression pattern among the functional units in each. This fact has made it difficult to discern the precise role of each ER in mediating the estrogen actions necessary to maintain homeostasis in the HPG axis. However, the generation of mice lacking one or both receptors provides the ideal tools for such investigations. Herein, we have thoroughly characterized the basic function of the HPG axis in females of all three lines of ERKO mice in anticipation that such a comparison may elucidate the contribution of each ER form in female reproductive endocrinology. Overall, our results indicate that the most overwhelming phenotypes follow the loss of ER{alpha}. However, it is also apparent that the manifestation of certain {alpha}ERKO phenotypes may indeed depend on the preservation of ERß functions as animals lacking both receptors often exhibit a very different phenotype.

Hypothalamic-Pituitary Function in the ERKO Female
The severe phenotypes exhibited by the {alpha}ERKO females, in contrast to the lack of comparable phenotypes in the ßERKO, indicate that ER{alpha} 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{alpha} and not ERß mediates estrogen regulation of Prl gene expression in the pituitary agrees with a previous report in the {alpha}ERKO (15) and now extends these findings to the {alpha}ßERKO female. Our observation that reduced Prl expression in the pituitary after either ovariectomy or the loss of functional ER{alpha} was not reflected in plasma PRL levels (Table 2Go) 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{alpha}, as only those mice lacking functional ER{alpha} 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 {alpha}ERKO females exhibited a comparable response to GnRH antagonist treatment indicates that the loss of ER{alpha} 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 {alpha}ERKO and {alpha}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 {alpha}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{alpha}, 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 {alpha}ERKO and {alpha}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-{alpha} (Inha), inhibin-ßA (Inhba), and inhibin-ßB (Inhbb), such that the possible combinations result in two potential forms of inhibin ({alpha}-ßA, {alpha}-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. 6Go). 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. 6AGo), 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{alpha} 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, {alpha}ERKO and {alpha}ßERKO females exhibited significant increases in Gnrhr expression, indicating that GnRH pulsatility is not only preserved after ER{alpha} 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{alpha} null mice vs. those lacking estradiol due to ovariectomy, as increased GnRH-R levels in the {alpha}ERKO and {alpha}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-{alpha} (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{alpha} 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 {alpha}ERKO hypothalamus (27, 28), our findings provide little indication as to its possible function, as only females lacking ER{alpha} exhibited aberrant GnRH and LH secretion. However, the increased serum LH observed in {alpha}ßERKO females relative to {alpha}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{alpha}, 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{alpha} 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{alpha} 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 {alpha}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 {alpha}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 {alpha}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{alpha}-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 {alpha}ERKO ovaries suggests ER{alpha} 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 {alpha}ERKO ovaries, indicating these phenotypes are subordinate to the loss of ER{alpha} 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 {alpha}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 {alpha}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 {alpha}ERKO ovaries (41). The return of Cyp19 expression to normal after the loss of both ERs in the {alpha}ßERKO ovary further supports this possibility. It is also plausible that the elevated androgens in the {alpha}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 {alpha}ERKO females did slightly reduce Cyp19 expression but was not nearly as effective as the GnRH antagonist. Lastly, increased steroidogenic enzyme expression in {alpha}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 {alpha}ßERKO females did not exhibit increased Cyp17 or Cyp19 expression, or the respective steroid end products, despite exhibiting a gonadotropin profile comparable to the {alpha}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 {alpha}ßERKO females (47). Adult {alpha}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 {alpha}ERKO-like increase in Cyp19 expression in the {alpha}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 {alpha}ßERKO ovary, it is also surprising that no {alpha}ERKO-like increase in Cyp17 expression was observed, despite these females possessing plasma LH levels that were more than 2-fold higher than {alpha}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 {alpha}ßERKO ovaries.

The most novel finding was the discovery of Hsd17b3 gene expression in the ovaries of {alpha}ERKO and {alpha}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. 3Go). Therefore, our finding of Hsd17b3 expression in {alpha}ERKO and {alpha}ßERKO ovaries at levels comparable to those in wild-type testes was quite surprising. Foremost, these data indicate that elevated plasma testosterone levels in {alpha}ERKO and {alpha}ßERKO females are due to an active synthesis within the ovaries and suggest that ER{alpha} may be involved in repressing Hsd17b3 expression in the wild-type ovary. These findings also extend previous descriptions of the {alpha}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 {alpha}ERKO ovary. Since assays to localize the Hsd17b3 expression in the {alpha}ERKO and {alpha}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 {alpha}ERKO and {alpha}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 {alpha}ERKO and {alpha}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{alpha}. The influence of increased androgens on Hsd17b3 expression was also apparent as flutamide treatment of {alpha}ERKO females significantly reduced expression, although this treatment proved not as effective as the GnRH antagonist.

Lastly, there are multiple similarities between the {alpha}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 {alpha}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 {alpha}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 {alpha}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 {alpha}ERKO female include obesity and insulin resistance (62), both of which are common in human PCOS (63). In fairness, one major difference is that {alpha}ERKO females do not exhibit the thickened outer capsule that is characteristic of human PCOS ovaries (63).

In summary, our findings indicate that ER{alpha} 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 {alpha}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 {alpha}ERKO ovary and provide further evidence that the most dramatic ovarian phenotypes in the {alpha}ERKO are due to chronic LH stimulation and not inherent to the loss of intraovarian ER{alpha} function. Interestingly, the manifestations of LH hyperstimulation in the {alpha}ERKO ovary may, in fact, depend on the presence of functional ERß as {alpha}ßERKO females exhibit a similar gonadotropin profile but lack consistent evidence of enhanced estradiol synthesis. The discovery of ectopic Hsd17b3 expression in {alpha}ERKO and {alpha}ßERKO ovaries extends the sex reversal phenotype in the {alpha}ßERKO ovary to now include an endocrine phenotype and marks the first such description of a similar phenotype in {alpha}ERKO ovaries.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation and Treatment of Animals
All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were preapproved by the National Institute of Enviromental Health Sciences Committee on Animal Care and Use. All animals were maintained in plastic cages under a 12-h light,12-h dark schedule in a temperature-controlled room (21–22 C) and fed NIH 31 mouse chow and fresh water ad libutum. The gene-targeting scheme used for the generation of each ERKO line has been described previously (64, 65). Mice were generated via heterozygous breeding pairs maintained in a continuous mating scheme. All animals were genotyped by PCR on DNA extracted from tail biopsy using the Puregene Genomic DNA extraction kit (Gentra Systems, Minneapolis, MN). PCR for detection of a wild-type or disrupted Esr1 gene used the following primers: exon 2, forward 5'-CGGTCTACGGCCAGTCGGGCATC, to intron 2, reverse 5'-CAGGCCTTACACAGCGGCCACCC (281-bp amplimer indicative of wild-type gene); neo, forward 5'-TGACCGCTTCCTCGTGCTTTAC (760-bp amplimer indicative of gene disruption). PCR for detection of a wild-type or disrupted Esr2 gene used the following primers: intron 1, forward 5'-TGGACTCACCACGTAGGCTC, to exon 2, reverse 5'-CATCCTTCACAGGACCAGACAC (356-bp amplimer indicative of wild-type gene); neo, forward 5'-GCAGCCTCTGTTCCACATACAC (404-bp amplimer indicative of gene disruption). All PCR results were evaluated by agarose gel electrophoresis.

All mice were 2–4 months of age with the exception of the {alpha}ßERKO mice used for gene expression assays, which ranged from 3–9 months of age. Where applicable, certain experiments included ovariectomized wild-type mice as a sex steroid-deficient model, in which case animals were ovariectomized 12–14 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 1200–1300 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 manufacturer’s 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 1Go). 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 manufacturer’s protocol. All blots were first probed for the experimental gene of interest, stripped according to the manufacturer’s 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 manufacturer’s protocol. The following probe sets were designed to allow for a single-tube RPA (see Table 1Go 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 1Go). The amount of total RNA used per assay ranged from 5–20 µg depending on the probe set and experiment. Hybridization times ranged from 1–1.5 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-{alpha}]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 manufacturer’s 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 1Go). 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 manufacturer’s 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 manufacturer’s protocol. PCR reactions were prepared using 1 µl cDNA per reaction for each respective primer set (Table 1Go) 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 1Go) 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-{gamma}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 manufacturer’s 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 manufacturer’s protocol. All final assay samples were quantified using a Packard Multi-Prias 2 {gamma} counter (Packard Instruments, Downer’s 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 Levene’s test. In cases where data sets failed the Levene’s 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 Fisher’s protected least-significant differences post hoc test. All statistical analyses were carried out using Statview 4.0 Software (SAS Institute, Inc., Cary, NC).


    FOOTNOTES
 
Abbreviations: ER, Estrogen receptor; ERKO, ER knockout; FOH, functional ovarian hyperandrogenism; GnRH-R, GnRH receptor; HP, hypothalamic-pituitary; HPG, hypothalamic-pituitary-gonadal; HSD, hydroxysteroid dehydrogenase; PCOS, polycystic ovarian syndrome; PRL, prolactin; RPA, ribonuclease protection assay; SQ-RT-PCR, semiquantitative RT-PCR.

Received for publication December 2, 2002. Accepted for publication February 26, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177–199[Medline]
  2. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Medline]
  3. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C 1985 The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 6:371–399[Medline]
  4. Hsueh AJW, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707–724[Medline]
  5. Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL 2000 Detection of estrogen receptor-ß messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 141:3506–3509[Abstract/Free Full Text]
  6. Skynner MJ, Sim JA, Herbison AE 1999 Detection of estrogen receptor {alpha} and ß messenger ribonucleic acids in adult gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5201[Abstract/Free Full Text]
  7. Herbison AE, Pape JR 2001 New evidence for estrogen receptors in gonadotropin-releasing hormone neurons. Front Neuroendocrinol 22:292–308[CrossRef][Medline]
  8. Sar M, Welsch F 1999 Differential expression of estrogen receptor-ß and estrogen receptor-{alpha} in the rat ovary. Endocrinology 140:963–971[Abstract/Free Full Text]
  9. Sharma SC, Clemens JW, Pisarska MD, Richards JS 1999 Expression and function of estrogen receptor subtypes in granulosa cells: regulation by estradiol and forskolin. Endocrinology 140:4320–4334[Abstract/Free Full Text]
  10. Gore-Langton RE, Armstrong DT 1994 Follicular steroidogenesis and its control. In: Knobil E, Jeill JD, eds. The physiology of reproduction. New York: Raven Press; 571–627
  11. Andersson S, Moghrabi N 1997 Physiology and molecular genetics of 17ß-hydroxysteroid dehydrogenases. Steroids 62:143–147[CrossRef][Medline]
  12. Hinshelwood MM, Demeter-Arlotto M, Means GD, Simpson ER 1993 Molecular biology of genes encoding steroidogenic enzymes in the ovary. In: Adashi EY, Leung PCK, eds. The ovary. New York: Raven Press; 165–183
  13. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417[Abstract/Free Full Text]
  14. Couse JF, Bunch DO, Lindzey J, Schomberg DW, Korach KS 1999 Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-{alpha} knockout mouse. Endocrinology 140:5855–5865[Abstract/Free Full Text]
  15. Scully KM, Gleiberman AS, Lindzey J, Lubahn DB, Korach KS, Rosenfeld MG 1997 Role of estrogen receptor {alpha} in the anterior pituitary gland. Mol Endocrinol 11:674–681[Abstract/Free Full Text]
  16. Sinha YN, Wickes MA, Salocks CB, Vanderlaan WP 1979 Gonadal regulation of prolactin and growth hormone secretion in the mouse. Biol Reprod 21:473–481[Medline]
  17. Saade G, London DR, Lalloz MR, Clayton RN 1989 Regulation of LH subunit and prolactin mRNA by gonadal hormones in mice. J Mol Endocrinol 2:213–224[Abstract]
  18. Parkening TA, Collins TJ, Smith ER 1982 Plasma and pituitary concentrations of luteinizing hormone, follicle-stimulating hormone and prolactin in aged, ovariectomized CD-1 and C57BL/6 mice. Exp Gerontol 17:437–443[Medline]
  19. Wierman ME, Gharib SD, Wang C, LaRovere JM, Badger TM, Chin WW 1990 Divergent regulation of gonadotropin subunit mRNA levels by androgens in the female rat. Biol Reprod 43:191–195[Abstract]
  20. Britt KL, Kerr J, O’Donnell L, Jones MEE, Drummond AE, Davis SR, Simpson ER, Findley JK 2002 Estrogen regulates development of the somatic cell phenotype in the eutherian ovary. FASEB J 16:1389–1397[Abstract/Free Full Text]
  21. Woodruff TK, Mather JP 1995 Inhibin, activin and the female reproductive axis. Annu Rev Physiol 57:219–244[CrossRef][Medline]
  22. Corrigan AZ, Bilezikjian LM, Carroll RS, Bald LN, Schmelzer CH, Fendly BM, Mason AJ, Chin WW, Schwall RH, Vale W 1991 Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 128:1682–1684[Abstract]
  23. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone. Science 202:631–633[Medline]
  24. Weiss J, Cote CR, Jameson JL, Crowley Jr WF 1995 Homologous desensitization of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone secretion in vitro occurs within the duration of an endogenous GnRH pulse. Endocrinology 136:138–143[Abstract]
  25. Stanislaus D, Pinter JH, Janovick JA, Conn PM 1998 Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone. Mol Cell Endocrinol 144:1–10[CrossRef][Medline]
  26. Kaiser UB, Sabbagh E, Katzenellenbogen BS, Conn PM, Chin WW 1995 A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 92:12280–12284[Abstract]
  27. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER{alpha}-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
  28. Shughrue P, Scrimo P, Lane M, Askew R, Merchenthaler I 1997 The distribution of estrogen receptor-ß mRNA in forebrain regions of the estrogen receptor-{alpha} knockout mouse. Endocrinology 138:5649–5652[Abstract/Free Full Text]
  29. Cowley SM, Hoare S, Mosselman S, Parker MG 1997 Estrogen receptors {alpha} and ß form heterodimers on DNA. J Biol Chem 272:19858–19862[Abstract/Free Full Text]
  30. Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J-A 1997 Mouse estrogen receptor ß forms estrogen response element binding heterodimers with estrogen receptor {alpha}. Mol Endocrinol 11:1486–1496[Abstract/Free Full Text]
  31. Leung PC, Goff AK, Kennedy TG, Armstrong DT 1978 An intraovarian inhibitory action of estrogen on androgen production in vivo. Biol Reprod 19:641–647[Medline]
  32. Leung PC, Armstrong DT 1979 Estrogen treatment of immature rats inhibits ovarian androgen production in vitro. Endocrinology 104:1411–1417[Medline]
  33. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity of steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract]
  34. Zhang FP, Poutanen M, Wilbertz J, Huhtaniemi I 2001 Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol Endocrinol 15:172–183[Abstract/Free Full Text]
  35. Samuels LT, Uchikawa T, Zain-ul-Abedin M, Huseby RA 1969 Effect of diethylstilbestrol on enzymes of cryptochid mouse testes of Balb-c mice. Endocrinology 85:96–102[Medline]
  36. Samuels LT, Bussmann L, Matsumoto K, Huseby RA 1975 Organization of androgen biosynthesis in the testis. J Steroid Biochem 6:291–296[CrossRef][Medline]
  37. Onoda M, Hall PF 1981 Inhibition of testicular microsomal cytochrome P-450 (17{alpha}-hydroxylase/C-17,20-lyase) by estrogens. Endocrinology 109:763–767[Abstract]
  38. Banks PK, Meyer K, Brodie AM 1991 Regulation of ovarian steroid biosynthesis by estrogen during proestrus in the rat. Endocrinology 129:1295–1304[Abstract]
  39. Sakaue M, Ishimura R, Kurosawa S, Fukuzawa NH, Kurohmaru M, Hayashi Y, Tohyama C, Ohsako S 2002 Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat. J Vet Med Sci 64:107–113[CrossRef][Medline]
  40. Govoroun M, McMeel OM, Mecherouki H, Smith TJ, Guiguen Y 2001 17ß-Estradiol treatment decreases steroidogenic enzyme messenger ribonucleic acid levels in the rainbow trout testis. Endocrinology 142:1841–1848[Abstract/Free Full Text]
  41. Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar M, Mayo KE, Korach KS 1999 Targeted disruption of the estrogen receptor-{alpha} gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology 140:2733–2744[Abstract/Free Full Text]
  42. Tetsuka M, Hillier SG 1997 Differential regulation of aromatase and androgen receptor in granulosa cells. J Steroid Biochem Mol Biol 61:233–239[CrossRef][Medline]
  43. Tetsuka M, Milne M, Hillier SG 1998 Expression of oestrogen receptor isoforms in relation to enzymes of oestrogen synthesis in rat ovary. Mol Cell Endocrinol 141:29–35[CrossRef][Medline]
  44. Ghersevich S, Nokelainen P, Poutanen M, Orava M, Autio-Harmainen H, Rajaniemi H, Vihko R 1994 Rat 17ß-hydroxysteroid dehydrogenase type 1: primary structure and regulation of enzyme expression in rat ovary by diethylstilbestrol and gonadotropins in vivo. Endocrinology 135:1477–1487[Abstract]
  45. Ghersevich S, Poutanen M, Tapanainen J, Vihko R 1994 Hormonal regulation of rat 17ß-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor. Endocrinology 135:1963–1971[Abstract]
  46. Danilovich N, Babu PS, Xing W, Gerdes M, Krishnamurthy H, Sairam MR 2000 Estrogen deficiency, obesity, and skeletal abnormalities in follicle-stimulating hormone receptor knockout (FORKO) female mice. Endocrinology 141:4295–4308[Abstract/Free Full Text]
  47. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen receptors {alpha} and ß. Science 286:2328–2331[Abstract/Free Full Text]
  48. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M 2000 Effect of single and compound knockouts of estrogen receptors {alpha} (ER{alpha}) and ß (ERß) on mouse reproductive phenotypes. Development 127:4277–4291[Abstract/Free Full Text]
  49. Magoffin DA 2002 The ovarian androgen-producing cells: a 2001 perspective. Rev Endocr Metab Disord 3:47–53[CrossRef][Medline]
  50. Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM 1999 Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary. Mol Endocrinol 13:1018–1034[Abstract/Free Full Text]
  51. Sha J, Baker P, O’Shaughnessy PJ 1996 Both reductive forms of 17ß-hydroxysteroid dehydrogenase (types 1 and 3) are expressed during development in the mouse testis. Biochem Biophys Res Commun 222:90–94[CrossRef][Medline]
  52. Baker PJ, Sha JH, O’Shaughnessy PJ 1997 Localisation and regulation of 17ß-hydroxysteroid dehydrogenase type 3 mRNA during development in the mouse testis. Mol Cell Endocrinol 133:127–133[CrossRef][Medline]
  53. Mustonen MV, Poutanen MH, Isomaa VV, Vihko PT, Vihko RK 1997 Cloning of mouse 17ß-hydroxysteroid dehydrogenase type 2, and analysing expression of the mRNAs for types 1, 2, 3, 4 and 5 in mouse embryos and adult tissues. Biochem J 325:199–205[Medline]
  54. Tsai-Morris CH, Khanum A, Tang PZ, Dufau ML 1999 The rat 17ß-hydroxysteroid dehydrogenase type III: molecular cloning and gonadotropin regulation. Endocrinology 140:3534–3542[Abstract/Free Full Text]
  55. Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG, Jones ME, Simpson ER, Findlay JK 2000 An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene. Endocrinology 141:2614–2623[Abstract/Free Full Text]
  56. Barbieri RL, Gao X 1997 Presence of 17ß-hydroxysteroid dehydrogenase type 3 messenger ribonucleic acid transcript in an ovarian Sertoli-Leydig cell tumor. Fertil Steril 68:534–537[CrossRef][Medline]
  57. Risma KA, Clay CM, Nett TM, Wagner T, Yun J, Nilson JH 1995 Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci USA 92:1322–1326[Abstract]
  58. Rosenfeld RL 1997 Current concepts of polycystic ovary syndrome. Baillieres Clin Obstet Gynaecol 11:307–333[Medline]
  59. Nelson VL, Legro RS, Strauss III JF, McAllister JM 1999 Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol Endocrinol 13:946–957[Abstract/Free Full Text]
  60. Jakimiuk AJ, Weitsman SR, Navab A, Magoffin DA 2001 Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries. J Clin Endocrinol Metab 86:1318–1323[Abstract/Free Full Text]
  61. Nelson VL, Qin K, Rosenfield RL, Wood JR, Penning TM, Legro RS, Strauss III JF, McAllister JM 2001 The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome. J Clin Endocrinol Metab 86:5925–5933[Abstract/Free Full Text]
  62. Heine PA, Taylor JA, Iwamoto GA, Lubahn DB, Cooke PS 2000 Increased adipose tissue in male and female estrogen receptor-{alpha} knockout mice. Proc Natl Acad Sci USA 97:12729–12734[Abstract/Free Full Text]
  63. Carr BR 1998 Disorders of the ovaries and female reproductive tract. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR, eds. Williams textbook of endocrinology. Philadelphia: WB Saunders Co; 751–817
  64. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166[Abstract]
  65. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
  66. Linzer DIH, Talamantes F 1985 Nucleotide sequence of mouse prolactin and growth hormone mRNAs and expression of these mRNAs during pregnancy. J Biol Chem 260:9574–9579[Abstract/Free Full Text]