Chronic Hypersecretion of Luteinizing Hormone in Transgenic Mice Selectively Alters Responsiveness of the {alpha}-Subunit Gene to Gonadotropin-Releasing Hormone and Estrogens

Rula A. Abbud, Rebecca K. Ameduri, J. Sunil Rao, Terry M. Nett and John H. Nilson

Department of Pharmacology (R.A.A., R.K.A., J.H.N.) Department of Biostatistics (J.S.R.) Case Western Reserve University Cleveland, Ohio 44106
Animal Reproduction and Biotechnology Laboratory (T.M.N.) Colorado State University Fort Collins, Colorado 80523


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones can act either at the level of the hypothalamus or the pituitary to regulate gonadotropin subunit gene expression. However, their exact site of action remains controversial. Using the bovine gonadotropin {alpha}-subunit promoter linked to an expression cassette encoding the ß-subunit of LH, we have developed a transgenic mouse model where hypersecretion of LH occurs despite the presence of elevated ovarian steroids. We used this model to determine how hypersecretion of LH could occur when steroid levels are pathological. During transition from the neonatal period to adulthood, the endogenous LHß subunit gene becomes completely silent in these mice, whereas the {alpha}-directed transgene and endogenous {alpha}-subunit gene remain active. Interestingly, gonadectomy stimulates expression of the endogenous {alpha} and LHß subunit genes as well as the transgene; however, only the endogenous LHß gene retains responsiveness to 17ß-estradiol and GnRH. In contrast, LH levels remain responsive to negative regulation by androgen. Thus, {alpha}-subunit gene expression, as reflected by both the transgene and the endogenous gene, has become independent of GnRH regulation and, as a result, unresponsive to estradiol-negative feedback. This process is accompanied by a decrease in estrogen receptor {alpha} gene expression as well as an increase in the expression of transcription factors known to regulate the {alpha}-subunit promoter, such as cJun and P-LIM. These studies provide in vivo evidence that estrogen-negative feedback on {alpha} and LHß subunit gene expression requires GnRH input, reflecting an indirect mechanism of action of the steroid. In contrast, androgen suppresses {alpha}-subunit expression in both transgenic and nontransgenic mice. This suggests that androgens must regulate {alpha}-subunit promoter activity independently of GnRH. In addition to allowing the assessment of site of action of sex steroids on {alpha}-subunit gene expression, these studies also indicate that chronic exposure of the pituitary to LH-dependent ovarian hyperstimulation leads to a heretofore-undescribed pathological condition, whereby normal regulation of {alpha}, but not LHß, subunit gene expression becomes compromised.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of LH synthesis and secretion involves an integrated hypothalamic-pituitary-gonadal axis. Pulsatile release of GnRH from the hypothalamus stimulates synthesis and secretion of LH (1, 2, 3). In females, LH stimulates thecal cell production of androstenedione, which diffuses to granulosa cells and undergoes conversion to estrogen by aromatase. FSH controls aromatase activity and also induces appearance of LH receptors in granulosa cells, further augmenting production of estrogen (4).

There is little doubt that sustained levels of androgens or estrogens feed back to inhibit secretion of LH and suppress levels of {alpha} and LHß mRNAs both in animal models and in in vitro studies (see Refs. 2, 5, 6 for review). The site of steroid action, however, remains unknown. Numerous studies in rodents support a hypothalamic site of action for steroid feedback, namely regulation of GnRH secretion. Others propose that estrogens feed back at both the hypothalamus and pituitary, while androgens act only at the hypothalamus (5). At the level of the pituitary, estrogen appears to modulate GnRH receptors to alter the responsiveness of the pituitary to GnRH (5). Finally, many of the discrepancies noted between these studies may reflect significant species-specific variation. In primates and humans, direct action of steroid hormones at the level of the pituitary has been reported (5, 7). Thus, the site of steroid action in control of LH synthesis and secretion still remains controversial.

Genetic evidence suggests that steroid receptors are critical for controlling gonadotropin gene expression. Elevated levels of LH are found in Tfm mice that lack functional androgen receptors (ARs) (8). This is a male-limited genetic trait as AR resides on the X chromosome (9, 10), and LH hypersecretion only occurs with complete loss of AR. Similarly, estrogen receptor knockout (ERKO) studies reported by Korach and colleagues (11, 12, 13) provide new evidence for the importance of ERs in controlling LH secretion. Female ERKO mice secrete elevated levels of LH and develop cystic ovaries. While these observations provide evidence for the importance of androgen feedback in the male and estrogen feedback in the female, they can not delineate the exact site of action of the steroids.

If sex steroids act at the level of gonadotropes to regulate secretion of LH, then gonadotropin subunit promoters must be regarded as likely targets. Numerous reports of steroid suppression of gonadotropin gene expression further increase this likelihood (see Refs. 2, 5 for review). Indeed, we demonstrated recently that AR represses activity of the {alpha}-subunit promoter in {alpha}T3–1 cells through a mechanism that requires the DNA-binding domain of the receptor but not its direct binding to the 5'-flanking region (14, 15). In contrast to AR, we were unable to detect a high-affinity binding site for ER within the human {alpha}-subunit gene or to observe a negative transcriptional effect of ER in transfection assays (16). While another group observed binding of ER to a 29-bp region within the proximal {alpha}-subunit promoter (17), transcriptional regulation occurred only in the presence of thyroid hormone receptor in GH3 cells.

With regard to the LHß gene, Shupnik and Rosenzweig (18) reported a high affinity-binding site for ER between -1174/-1159 bp of the 5'-flanking region of the rat gene. Estrogen, however, conferred a positive, rather than negative, response when this element was tested for activity by transfection assays in GH3 cells. Subsequently, we observed that a truncated bovine LHß promoter (780 bp) directs expression of reporter genes specifically to gonadotropes in transgenic mice (19). This promoter also conferred positive responsiveness to GnRH and negative responsiveness to androgens and estrogens. Ironically, this steroid-responsive promoter lacked high-affinity binding sites for both AR and ER, suggesting that steroid receptors repress transcription of the bovine LHß gene, and possibly other mammalian LHß genes, through an indirect mechanism.

Recently, we constructed a new transgenic mouse model in which elevated serum levels of a chimeric LH heterodimer were maintained chronically without requiring multiple injections, pharmacological dosing, or artificial dampening of the hypothalamic-pituitary-gonadal axis (20, 21). The transgene contained a bovine {alpha}-subunit promoter fused to the coding region of a chimeric LHß subunit. The {alpha}-subunit promoter ensured that transgene expression occurred only in gonadotropes (20, 22, 23). The LHß chimera contained the carboxyl-terminal peptide (CTP) of the human CG ß-subunit linked to the carboxyl terminus of bovine LHß. Addition of the CTP moiety increased the serum half-life of heterodimers containing the chimeric ß-subunit (20). The CTP-containing chimera was expressed only in gonadotropes of transgenic mice, causing elevated levels of LH. These mice were infertile, ovulated infrequently, maintained a prolonged luteal phase, and developed pathological ovarian changes including polycystic ovaries, marked enlargement of ovaries, and granulosa cell tumors (20, 21, 24). In addition, testosterone and estradiol (E2) levels were increased compared with nontransgenic littermates with an overall increase in the ratio of testosterone to E2 (20). In short, the biochemical and morphological features of these mice bear a striking resemblance to those of women with functional ovarian hyperandrogenism (25).

Our transgenic model demonstrates, for the first time, a direct association between abnormal secretion of LH, development of functional ovarian hyperandrogenism, and infertility. Since the transgene is the only variable in this animal model, expression of transgenic LH must be the initiating step for all subsequent pathological events. However, hypersecretion of LH occurred in female but not male mice. In female transgenic mice, the high levels of LH suggest that the gonadotropin genes, including the transgene, must manifest a resistance to steroid-negative feedback. Thus, this model provides a unique opportunity to explore developmentally how high levels of LH can be maintained in the presence of a chronically hyperstimulated ovary. The work reported herein reveals several unexpected findings that are relevant to both site of action of gonadal steroids and the dependency of their action on GnRH.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hypersecretion of LH Occurs Despite the Presence of Apparent Gonadal Feedback
To establish a frame of reference for understanding how hypersecretion of LH occurs in transgenic females in the presence of elevated gonadal steroids, we measured serum LH in intact and ovariectomized (OVX) animals. Mean serum levels of LH in intact transgenic mice are elevated compared with their nontransgenic counterparts (Fig. 1Go). In fact, levels of LH in transgenic females are comparable to those in OVX nontransgenic mice (Fig. 1Go). Transgenic mice, however, do not lack gonadal feedback, because serum levels of LH increase to even higher levels after ovariectomy. This suggests that the high levels of sex steroids in the intact transgenic animal are at least partially capable of suppressing LH levels.



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Figure 1. Hypersecretion of LH in Female Transgenic Mice Is Not Due to the Lack of Gonadal Feedback

Serum LH levels in female transgenic and nontransgenic (control) mice in the presence or absence of the gonads. Age-matched female transgenic (Intact, n = 5; OVX, n = 4) and nontransgenic (Intact, n = 7; OVX, n = 6) mice were killed in the morning hours at random stages of the estrous cycle. a, P < 0.05 when compared with Intact of the same genotype.

 
Partial responsiveness to gonadal feedback could reflect changes in gonadal regulation of LHß encoded by the transgene, the endogenous gene, or both. To begin to address these possibilities, we used an RNAse protection assay to measure the relative concentration of the endogenous mouse LHß (mLHß) and the bLHßCTP transgene-encoded mRNAs in total RNA prepared from individual mouse pituitaries. This assay allowed us to distinguish between the bLHßCTP transgene and endogenous mLHß gene. In intact transgenic females, bLHßCTP mRNA is readily detectable, whereas the endogenous mLHß mRNA is not discernible (Fig. 2Go). Upon gonadectomy, the endogenous mLHß mRNA becomes apparent and bLHßCTP mRNA appeared to increase even further. In contrast, endogenous mLHß mRNA is readily detectable in nontransgenic animals and increases after ovariectomy. These results indicate that serum LH in intact, transgenic adults is comprised of ß-subunit produced solely by the bLHßCTP transgene. Furthermore, whereas gonadal factors completely suppress the endogenous LHß gene in transgenic animals, they only partially suppress the bLHßCTP transgene. Since the LHß-encoding transgene is linked to the heterologous {alpha}-subunit promoter, we posit that the {alpha} and LHß promoters respond differentially to gonadal feedback.



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Figure 2. Reversible Suppression of Endogenous Mouse LHß mRNA Levels in Transgenic Mice

RNAse protection assays were performed to detect mLHß, transgene, and actin mRNA levels in individual mouse pituitaries from transgenic and nontransgenic mice. Quantitative analysis of mLHß and transgene mRNA expressed relative to actin in transgenic and nontransgenic (Control), OVX, and ovarian-intact (Intact) pituitaries. aP < 0.05 when compared with Intact. bP < 0.05 when compared with Control.

 
Partial Escape of Transgene Expression from Suppression by Elevated Ovarian Steroids Becomes Evident at the Onset of Puberty in Female Transgenic Mice
We reported previously that hypersecretion of LH occurred as early as 14 days of age even though androgens and estrogens were at levels higher than those seen in an adult nontransgenic mouse (21). To study the relative contribution of the bLHßCTP transgene and the endogenous LHß gene to the total LHß mRNA pool during early pre- and postnatal development in female transgenic mice, we used both double-label in situ hybridization and RNAse protection assays. This analysis further confirmed that the transgene is expressed specifically in cells that express the {alpha}-subunit of LH (data not shown).

Expression of the transgene was detected with an 35S-labeled probe, whereas a digoxigenin-labeled probe was used to assess expression of the endogenous LHß gene. Expression of endogenous mLHß mRNA was observed from embryonic day 19 up to postnatal day 14 in transgenic animals (Fig. 3Go, panels B–D) and was similar to that observed in nontransgenic littermates (data not shown). Activity of the bLHßCTP transgene was also detected in the pituitary at these early stages of development (Fig. 3AGo and data not shown). In contrast, little staining for endogenous mLHß mRNA was observed in pituitary sections from adult female transgenic mice, especially when compared with nontransgenic siblings (Fig. 3Go, E and F). As expected, adult transgenic pituitaries expressed high levels of the transgene (data not shown).



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Figure 3. Endogenous mLHß Gene Expression Becomes Suppressed after Puberty

A, Pattern of transgene expression in the anterior pituitary at embryonic day 19. Dark field image showing the results from in situ hybridization using 35S-labeled transgene probe on coronal sections from an embryo at embryonic day 19. The transgenic signal appears as silver grains in the developing anterior pituitary, Magnification, x40. B–F, Endogenous mLHß gene expression in transgenic female mice at E19 (B, x40), P-1 (C, x40), P-14 (D, x40), and adult (E, x400) stages of development. The mLHß signal appears as a dark stain using digoxigenin-labeled mLHß riboprobe for in situ hybridization. Note that endogenous gene expression becomes completely suppressed in the adult pituitary. F, Pattern of mLHß gene expression in adult nontransgenic mouse pituitaries using in situ hybridization with digoxigenin- labeled mLHß riboprobe.

 
To determine when endogenous LHß gene expression becomes suppressed, we performed RNAse protection assays on single pituitaries from animals killed at 3, 4, and 6 weeks of age. As shown in Fig. 4Go, endogenous mLHß mRNA levels progressively declined, becoming completely suppressed by 6 weeks of age. At 3 weeks of age both the transgene and endogenous gene contribute equally to pituitary LHß mRNA, coincident with precocious follicular maturation as evidenced by early antral formation (24). Complete suppression of endogenous gene expression occurs at 6 weeks of age, a time normally associated with the onset of puberty. In nontransgenic animals, gonadal regulation of pituitary gonadotropin levels becomes fully mature at puberty, around 6 weeks of age (26). This is clearly not the case for the transgenic mice because the transgene is demonstrating partial gonadal feedback insensitivity. Thus, secreted LH remains elevated and ultimately causes the demise of ovarian function.



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Figure 4. Endogenous mLHß but Not bLHßCTP Transgene Expression Becomes Suppressed by 6 Weeks of Age

Pattern of transgene and mLHß gene expression expressed relative to actin in female transgenic pituitaries at 3 (n = 3), 4 (n = 6), and 6 (n = 7) weeks of age as determined by RNAse protecion assay. a P < 0.05 when compared with 3 weeks.

 
Selective Resistance to Estrogen-Negative Feedback Causes Hypersecretion of LH
To further understand how transgene expression becomes partially insensitive to gonadal feedback in adult females, we evaluated levels of serum LH in OVX animals before and after selective steroid hormone replacement. Figure 5Go shows that replacement with 5{alpha}-dihydrotestosterone (DHT), a nonaromatizable analog of T, suppressed LH levels to the same extent in both transgenic and nontransgenic female mice (mean OVX transgenic: 64.6 ± 1.2 ng/ml; OVX nontransgenic: 5.3 ± 0.9 ng/ml; OVX + DHT transgenic: 9.9 ± 4.6 ng/ml; OVX + DHT nontransgenic: 1.3 ± 0.3 ng/ml). Indeed, mean serum LH levels in DHT-treated females were indistinguishable from the levels observed in intact female transgenic mice (data not shown). This suggests that androgens may be the primary agent for gonadal feedback in transgenic females. In further support of this notion, 17ß-estradiol suppressed LH levels less effectively in transgenic mice than in nontransgenic littermates (mean OVX transgenic: 68.3 ± 6.6 ng/ml; OVX nontransgenic: 6.5 ± 1.4 ng/ml; OVX + E transgenic: 25.8 ± 2.28 ng/ml; nontransgenic: 0.6±0.1 ng/ml). Therefore, hypersecretion of LH appears to reflect transcriptional resistance to estrogen-negative feedback. Since the bLHßCTP transgene is the primary source of secreted LH in intact females, it follows that the {alpha}-subunit promoter must be the target of this partial resistance.



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Figure 5. Suppression of the Postovariectomy Rise in LH by E2, but Not DHT, Is Compromised in Female Transgenic Mice

Serum levels of LH are expressed relative to the levels obtained after ovariectomy in animals implanted with either E2 or DHT pellets. Both transgenic and nontransgenic animals were OVX for several weeks before administration of the steroid implants. OVX + DHT, Nontransgenic: n = 5; OVX + DHT, transgenic: n = 6; OVX + E2, nontransgenic: n = 6, OVX + E2, transgenic: n = 6. a P < 0.05 when compared with nontransgenic.

 
The rise in serum LH in transgenic animals after ovariectomy results from contributions by both the endogenous LHß gene and the chimeric bLHßCTP transgene. To determine whether altered responsiveness to E2 is a unique property of the transgene, we measured the effect of E2 implants after ovariectomy on mLHß and bLHßCTP transgene mRNA levels. Whereas both endogenous mLHß and bLHßCTP transgene mRNA levels increased after ovariectomy, only the endogenous mLHß gene was suppressed by 17ß-estradiol implants (Fig. 6Go). These results suggest that the hypersecretion of LH in adult female transgenic mice could be attributed to resistance of the transgene to negative feedback by E2. Interestingly, although the transgene remains responsive to androgen regulation, androgens in the intact transgenic female alone are insufficient to prevent LH hypersecretion.



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Figure 6. The Transgene Is Unresponsive to E2-Negative Feedback in Female Transgenic Mice

mLHß and transgene mRNA levels in intact (n = 9), OVX (n = 13), and OVX + E (n = 9) were determined by RNAse protection assay, normalized relative to actin mRNA levels, and expressed relative to OVX levels. a P < 0.05 when compared with OVX, b P > 0.05 when compared with OVX.

 
Expression of the Endogenous {alpha}-Subunit Gene Also Becomes Resistant to Estrogen-Negative Feedback in Response to Chronic LH Hypersecretion
The inability of E2 to regulate transgene expression is surprising in light of previous work demonstrating that this same bovine {alpha}-subunit promoter responds to estrogen-negative feedback when linked to chloramphenicol acetyltransferase (CAT) in transgenic mice (14, 16). This suggests that chronic hypersecretion of LH causes the truncated {alpha}-subunit promoter to become unresponsive to estrogen-negative feedback. To determine whether this was a unique property of the transgene or whether a fundamental change had occurred in the normal endocrine feedback loop, we assessed the ability of E2 to regulate the endogenous {alpha}-subunit gene. As shown in Fig. 7Go, expression of the endogenous mouse {alpha}-subunit mRNA is compromised in transgenic mice when compared with nontransgenic littermates. While it is still responsive to ovariectomy, it fails to respond to E2 implants. Therefore, the endogenous mouse {alpha}-subunit promoter becomes dysregulated in the same manner as the transgene. These findings suggest that a pathological change has occurred that affects appropriate regulation of {alpha} but not LHß subunit gene expression.



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Figure 7. The Endogenous {alpha}-Subunit Becomes Unresponsive to E2-Negative Feedback

Mouse {alpha}mRNA was determined by RNAse protection assay and expressed relative to actin. Data shown are from nontransgenic (Control) and transgenic, intact (Control, n = 5; Transgenic, n = 6), OVX (Control, n = 5; Transgenic, n = 3), and OVX + E (Control, n = 5; Transgenic, n = 4) mice. aP < 0.05 when compared with OVX. bP > 0.05 when compared with OVX.

 
The Loss of {alpha}-Subunit Responsiveness to E2 Correlates with a Loss of Regulation by GnRH
Estrogen-negative feedback on gonadotropin gene expression can occur either at the level of the hypothalamus, pituitary, or both (1, 2). If the repressive effect of estrogen on {alpha}-subunit promoter activity occurs predominately at the hypothalamus by diminishing production of GnRH, then administration of a GnRH antagonist should block the postcastration rise in {alpha}-subunit mRNA. Surprisingly, treatment of OVX transgenic females with antide, a GnRH antagonist, had no impact on transgene mRNA, whereas it was much more effective in blocking the postcastration rise in endogenous LHß mRNA (Fig. 8Go). This suggests that expression of the transgene is independent of GnRH and its postovariectomy rise is due to regulation at the level of the pituitary rather than the hypothalamus. This independence of GnRH regulation is selective for the {alpha}-subunit promoter since the endogenous LHß mRNA retains responsiveness to antide. Furthermore, GnRH levels must be quite low in intact transgenic females since treatment with antide had no impact on serum levels of LH (data not shown). However, this treatment had no measurable effect in nontransgenic randomly cycling females because circulating levels of LH in these animals are at the limits of detection by RIA. Consequently, treatment with antide has a measurable effect only when levels of LH are first elevated by ovariectomy, which causes a dramatic increase in GnRH. The inability of antide to block the postcastration rise in transgenic bLHßCTP mRNA, but not endogenous LHß mRNA, provides evidence for the reprogramming of the responsiveness of the {alpha}-subunit promoter. In addtion, it indicates that the {alpha}-subunit promoter retains appreciable activity even in this environment of low GnRH. More importantly, since E2 fails to suppress transgene activity in this altered endocrine milieu, the steroid must normally exert its effect on the {alpha}-subunit promoter through an indirect mechanism that involves regulation of GnRH.



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Figure 8. Transgene Expression Becomes Independent of GnRH in Transgenic Mice

Transgene and endogenous (mLHß) mRNA levels expressed relative to actin in transgenic and nontransgenic (Control) mice that were OVX and treated either with saline (ovx + saline) or antide (ovx + antide). n = 4–7 animals per treatment group. aP < 0.05 when compared with OVX + saline. b P > 0.05 when compared with OVX + saline.

 
Increased Expression of trans-Activators of the {alpha}-Subunit Promoter in the Pituitaries of Female Transgenic Mice: Possible Mechanism of {alpha}-Subunit Autonomy from GnRH and E2
To further understand how {alpha} but not LHß becomes independent of GnRH regulation and refractory to estrogen-negative feedback, we measured the expression of GnRH (GnRH-R), androgen (AR), and the {alpha}-isoform of estrogen (ER) receptors using reverse Northern assays. We chose to study the {alpha}- but not ß-isoform of ER because ERß does not appear to be expressed in the pituitary (27). In addition, ERKO mice, which express ERß but not ER{alpha}, have elevated levels of LH despite high levels of E2 (13). Thus, E2-negative feedback on LH appears to be mediated by ER{alpha} rather than ERß. In addition, we examined the expression pattern of several transcription factors that are known to regulate both {alpha} and LHß subunit gene expression. These include steroidogenic factor-1 (SF-1) (28, 29, 30); P-Otx/Ptx1 (31, 32); P-LIM (33, 34), cJun (35), and cAMP response element binding protein (CREB) (35). SF-1 and P-Otx/Ptx1 act synergistically to regulate the LHß promoter (31). P-Otx/Ptx1 also acts in synergy with another factor, P-LIM, to regulate {alpha}-subunit gene expression (32). In the pituitary, expression of the {alpha}-subunit promoter in the transgene and endogenous {alpha}-subunit gene depends on a variant cAMP response element sequence (CRE). CREB binds this sequence in pituitary cell lines but at a low affinity. However, cJun and ATF-2 also bind this sequence but with high affinity in the pituitary (35). Furthermore, we analyzed the expression of the protooncogenes cFos, ras, and c-myc.

Statistical analysis of the reverse Northern data required inverse log transformation. Figure 9Go shows the difference between the inverse log net counts in transgenic and nontransgenic samples. Only three factors showed statistical difference in their expression pattern between transgenic and nontransgenic pituitaries. ER expression was significantly decreased, while cJun and P-LIM expression was increased in transgenic mouse pituitaries. Changes in the expression pattern of the other genes were not significantly different. Thus, chronic exposure to high levels of LH affected the expression of the ER, cJun, and P-LIM. These changes might be involved in the pathological events that alter regulation of the {alpha}-subunit promoter by GnRH and E2.



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Figure 9. Quantitative Analysis of Reverse Northern Slot-Blot Analysis Revealing Differential Expression of an Array of Genes in Transgenic and Nontransgenic Mice

Data were inverse log transformed and the difference between transgenic and nontransgenic values was plotted. aP < 0.05 statistical difference.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data reported herein indicate that chronic hypersecretion of LH renders the {alpha}-subunit promoter independent of GnRH and, as a result, unresponsive to estrogen-negative feedback. This same promoter appeared to be normally regulated by androgens since DHT suppressed the postcastration rise in LH in these animals. In contrast, the endogenous LHß gene remains fully regulated by GnRH and steroid hormones. It is not unexpected to see the two subunits of LH responding differently under these pathological conditions because both promoters have been previously reported to be differentially regulated by GnRH (36, 37, 38). However, it is surprising that the {alpha}-subunit promoter has become unresponsive to the postovariectomy rise in GnRH and, as a result, unresponsive to E2-negative feedback.

With chronic exposure to elevated LH, expression of the {alpha} and LHß genes vary dramatically in their requirement for GnRH. Expression of the LHß gene depends almost entirely on GnRH, whereas activity of the {alpha}-subunit promoter has a significant component of independence. From a teleological point of view, this makes sense since production of the defining glycoprotein hormones for at least three cell types (gonadotropes, thyrotropes, and trophoblasts) requires expression of the {alpha}-subunit gene. Since only one of these cell types, gonadotropes, has a GnRH-R with clearly defined functional significance, it follows that activity of the {alpha}-subunit promoter should only partially depend on GnRH. In contrast, expression of the LHß subunit gene, one of the defining characteristics of the gonadotrope, appears to fundamentally require appropriate, sustained, pulses of GnRH.

Our strongest evidence for the differential requirement of {alpha} and LHß gene expression for GnRH comes from the antide treatment studies carried out in OVX females (Fig. 8Go). Castration removes steroids and releases the hypothalamus from their tonic suppressive effect. The ability of antide to block the postcastration rise in LHß mRNA provides indirect evidence for both the presence of, and requirement for, GnRH. The inability of the transgenic {alpha}-subunit promoter to respond to the antide paradigm suggests that the {alpha}-subunit has significant residual activity in the presence of high steroids. Since GnRH release is likely suppressed under these conditions, the {alpha}-subunit promoter must function in the absence of GnRH and has lost its ability to respond to its restoration upon castration.

Loss of {alpha}-subunit promoter responsiveness to GnRH and E2 in transgenic mice is probably mediated by the pathological effects of exposure to high levels of LH early in life. In previous transgenic studies with a CAT reporter gene, we found that the same bovine {alpha}-subunit promoter used in this study confers responsiveness to both GnRH and E2 (16). Thus, a fundamental change has occurred in these animals as a result of placing LHß subunit gene expression under control of the {alpha}-subunit promoter. This change is probably caused by a complex sequelae of events. During the infantile period, both the bLHßCTP transgene and endogenous LHß gene are expressed at a time when androgens and estrogens are elevated over that seen in nontransgenic control mice (21). Repression of endogenous LHß gene expression was evident during the prepubertal period and became complete at puberty, which is precocious in these mice. This is most likely caused by a reduction of GnRH due to excessive steroid levels. However, coincident with the suppression of the mLHß subunit gene, the endogenous {alpha}-subunit gene and the transgene develop independence of GnRH and insensitivity to E2.

It is apparent from these studies that the use of the {alpha}-subunit promoter to target expression of the chimeric LHß subunit was key for the success of our LH hypersecretion model. Exposure to elevated levels of LH results not only in the escape of the transgenic {alpha}-subunit promoter from GnRH and E2 regulation, but also alters the regulation of the endogenous {alpha}-subunit gene. Thus, the observed effects on the {alpha}-subunit promoter are due to chronically elevated levels of LH rather than some intrinsic property unique to the transgene. The LHß subunit promoter would not have been able to recapitulate this phenotype, since it retained responsiveness to E2 and GnRH.

Loss of GnRH responsiveness must have occurred through reprogramming of key signal transduction cascades. This may involve concerted changes in several transcription factors, including steroid hormone receptors, that normally regulate the {alpha}-subunit promoter. These changes appear permanent, suggesting that activity of the promoter is reprogrammed either early in development or later after the onset of puberty. As a first approach to understanding the molecular mechanisms underlying altered {alpha}-subunit promoter regulation, we focused on transcription factors that are known to be expressed in the pituitary and regulate gonadotropin gene expression (Fig. 9Go). It is clear that chronic hypersecretion of LH causes changes in ER, cJun, and P-LIM gene expression. These changes could be occurring in any cell type in the pituitary. However, we can speculate on the significance of these changes since all three genes are known to be involved in the regulation of gonadotropin subunit gene expression. P-LIM and cJun are known to regulate basal expression of the {alpha}-subunit gene by binding to cognate sites in the proximal promoter region (32). The site of P-LIM binding in the mouse and human {alpha}-subunit promoters, which is called PGBE, has been shown to mediate GnRH responsiveness. Furthermore, GnRH stimulates the JNK-signaling pathways in cell lines of the gonadotrope lineage (39). Thus, increased expression of both cJun and P-LIM may convey significant promoter activity in the transgenic mouse pituitaries in the absence of GnRH. In addition, the decrease in ER gene expression may reflect an attempt to up-regulate endogenous mLHß gene expression. While the functional impact of these changes awaits further analysis, it is plausible that changes in their expression pattern may allow a compensatory mechanism for the {alpha}-subunit promoter to lose E2 responsiveness and retain appreciable activity even in the absence of GnRH.

One of the strengths of our pathophysiological model of LH hypersecretion is that the absence of GnRH permitted clear identification of the site of action regarding estrogen suppression of {alpha}-subunit promoter activity. In female transgenic mice, GnRH input appears to be suppressed, presumably by the high levels of estrogen. Thus, in the absence of GnRH, estrogen has no effect on residual levels of {alpha}-subunit because there is no pituitary mechanism that enables estrogen to regulate activity of the {alpha}-subunit promoter. Indeed, in previous studies we demonstrated that a larger human {alpha}-subunit promoter lacks a high affinity-binding site for ER and cannot respond to estrogen in cotransfection studies with human ER (16). Like the bovine promoter (16, 23), this human {alpha}-subunit promoter also confers responsiveness to GnRH and estrogen in a transgenic setting. Taken together, these studies provide compelling evidence that estrogen suppresses activity of the {alpha}-subunit promoter through an indirect mechanism that occurs at a distal site of action, most likely the hypothalamus.

Synthesis of LH involves expression of the {alpha} and LHß subunit genes. For the {alpha}-subunit, our data strongly suggest that estrogen exerts its action solely at the level of the hypothalamus, where it regulates production of GnRH. In contrast, activity of the LHß promoter is completely dependent on continuous pulses of GnRH. With no GnRH (due to hypothalamic estrogen-negative feedback), the endogenous LHß gene is completely suppressed in female transgenic mice. Because the LHß promoter is silent, we cannot rule out additional effects of estrogen that utilize transcriptional pathways within a gonadotrope. What we can say is that for promoters such as the {alpha}-subunit, where appreciable activity remains when GnRH is absent, resistance to estrogen-negative feedback is most likely linked directly to escape from GnRH regulation. For promoters like LHß that are completely dependent on GnRH, there may be additional sites of estrogen action that include effects on GnRH receptor and components of its downstream signaling pathway.

Thus, when considering resistance of LH to steroid-negative feedback, there are two primary genes ({alpha} and LHß) and two cellular sites of action (gonadotropes and GnRH neurons) that together present a combinatorial array of targets that, if any single one is affected, could result in hypersecretion of LH. Our work singles out one mechanism that illustrates how the GnRH independence of {alpha}-subunit allows appreciable expression to occur when higher than normal concentrations of E2 abound. This finding provides an important paradigm for understanding how hypersecretion of LH could occur in women. Our observations in mice have been recently confirmed in women with polycystic ovary syndrome who appear to have elevated levels of LH as a result of resistance to steroid-negative feedback (40). However, the mechanism involved in the escape of LH from steroid-negative feedback remains unclear. Since ovarian hyperstimulation will suppress secretion of GnRH, either GnRH neurons must become resistant to steroid-negative feedback or the dependence of the LHß promoter on GnRH must be relieved presumably through alterations in gonadotropes that give the promoter some transcriptional autonomy.

In contrast to estrogen, androgens appear to suppress activity of the {alpha}- subunit promoter through a direct mechanism. Thus, androgen action can be distinguished from estrogen action by its ability to regulate the {alpha}-subunit promoter in the absence of GnRH. Our earlier AR cotransfection studies indicated that androgens repress activity of the human {alpha}-subunit promoter through a mechanism that does not require binding of AR directly to DNA (14, 15). Instead, AR responsiveness maps to two elements required for {alpha}-subunit promoter basal activity [CRE and {alpha}BE; (41)] suggesting some form of transcriptional interference. In the end, while chronic hypersecretion of LH renders the {alpha}-subunit promoter refractory to GnRH and estrogen, responsiveness to androgen remains intact. Our next challenge will be to map the complete pathway that permits androgen regulation of the {alpha}-subunit promoter independent of GnRH.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Female transgenic mice carrying the bLHßCTP transgene were obtained by breeding male transgenic mice with CF-1 female nontransgenic mice and were at least seven generations from the founder animal. Mice were genotyped by PCR of tail DNA using primers specific for the {alpha}-subunit promoter and the bovine LHß subunit gene as reported earlier (20). In addition, the sex of the embryos used in these studies was determined by PCR using primers for Sry: 5'-GAGAGCATGGAGGGCAT-3' and 5'-CCACTCCTCTGTGACACT-3'. All mice were housed under controlled light (12 h light, 12 h dark) and temperature (25 C) conditions. All experimental procedures were approved by the Case Western Reserve University Institutional Animal Care and Use Committee.

Developmental Studies
Female transgenic mice and nontransgenic littermates were killed by decapitation at different stages of development: embryonic day 19 (n = 2 per group); postnatal day 1 (n = 2 per group); postnatal day 14 (n = 2 per group); 3 weeks of age (n = 3); 4 weeks of age (n = 6); and 6 weeks of age (n = 7). Pituitaries or heads (for younger animals) were immediately frozen and stored at -80 C until used in RNAse protection assays or in situ hybridization. Ovaries were inspected for obvious phenotypes. Trunk blood was collected and serum LH levels were determined by a previously validated RIA (20).

Treatment Paradigms
Adult female transgenic and nontransgenic littermates were OVX under Avertin (1:1 Tribromoethanol:tertiary-amylalcohol stock, diluted 1:40 in normal saline) anesthesia. Orbital sinus blood samples were obtained under anesthesia before OVX or treatment. The effect of estrogen-negative feedback was tested in chronically OVX mice (2–4 weeks) implanted subcutaneously with 17ß-estradiol constant-release pellets (0.25 mg/pellet; Innovative Research of America, Toledo, OH) for 2–3 weeks (13–15 animals per group). This pellet results in serum E2 levels of approximately 100 pg/ml (19). DHT pellets (7.5 mg/pellet, Innovative Research of America) were similarly implanted after ovariectomy to obtain a serum concentration of approximately 5 ng/ml (5–6 animals per group). Control animals were implanted with placebo pellets. For the ovariectomy/antide treatment paradigm, we used the same protocol that has been previously used in our laboratory with subcutaneous injections of antide (60 µg in 20% propylene glycol in normal saline per animal, once every other day for 10 days; n = 4–7 animals per group) (19). At the end of each treatment, animals were killed by decapitation and trunk blood was collected. Pituitaries were immediately frozen and stored at -80 C. Total RNA was isolated using the Trizol method (Life Technologies, Inc., Gaithersburg, MD).

Riboprobes Used For Both in Situ Hybridization and RNAse Protection Assay
For double-label in situ hybridization, the endogenous {alpha} and LHß probes were labeled with digoxigenin and the bLHßCTP probe was 35S labeled. The mouse {alpha} cDNA was obtained from Dr. David Gordon, University of Colorado Health Science Center (Denver, CO). It is a 460-bp PstI insert in pGEM3Zf+ (Promega Corp., Madison, WI). After linearization with EcoRI the riboprobe was synthesized using SP6 RNA Polymerase in the presence of 80% Digoxigenin-11-UTP (Roche Molecular Biochemicals, Indianapolis, IN). The mouse LHß cDNA is a 250-bp fragment that was cloned in the SmaI site of BSK- (Stratagene, La Jolla, CA) (42). Antisense digoxigenin-labeled riboprobe was generated after linearization of the plasmid with HindIII and using T7 RNA Polymerase. bLHßCTP cDNA (520bp) inserted in the KpnI/BamHI site of BKS- was used for transgene probe synthesis. The transgene probe was generated after linearization with StuI and using T7 RNA Polymerase. All modifying enzymes were obtained from Life Technologies, Inc..

RNase Protection Assay and in Situ Hybridization
For the RNAse protection assays, 32P-labeled riboprobes for mLHß, m{alpha}, and bLHßCTP were synthesized using the same cDNA templates mentioned above. As a control, a riboprobe for mouse ß-actin was also generated. The mouse ß-actin template is obtained by digesting the pTRI-B-Actin-Mouse vector (Ambion, Inc., Austin, TX) with DdeI. The mLHß, bLHßCTP, and actin probes are 250 bp, 387 bp, and 160 bp, respectively, allowing detection of each RNA in the same sample. For m{alpha}, four protected bands were observed. The signal from all four bands with background subtracted was expressed relative to actin.

RNAse protection assays were performed on total RNA using the RPAII kit (Ambion, Inc.). Total RNA from individual pituitaries was denatured and coprecipitated with 32P-labeled riboprobes for mLHß, bLHßCTP, and actin (7.3 x 104 cpm of each probe). After overnight hybridization at 42 C, samples were treated with RNAse A/TI (1:100 dilution) and precipitated according to kit instructions. Pellets were resuspended in nondenaturing loading buffer (16% sucrose, 0.05% bromophenol blue, 0.05% xylene cyanol) and run on nondenaturing 4.5% polyacrylamide gels. Gels were dried and exposed to x-ray film overnight at -80 C. A standard curve was generated for each probe using in vitro transcribed sense RNA (data not shown). Quantitative analysis of the signal from each band was performed using the Ambis Radioanalytical Imaging system (AMBIS, Inc., San Diego, CA). The background was subtracted from the signal for each band. Data were expressed relative to actin as mean values ± SEM, and statistical analysis was performed using the unpaired Student’s t test with unequal variance.

Double-label in situ hybridization, using digoxigenin-labeled probe for mLHß and 35S-labeled probe for bLHßCTP, was performed on fresh frozen 10-µm sections as described earlier (42, 43). For all the RNA work, sense probes were used to control for specificity of the hybridization signal.

Reverse Northern Assays
Reverse Northern slot blot assays (n = 3) were performed using a modified version of previously published protocols (44, 45, 46). The cDNA clones used in this experiment were as follows: human ER (47), mouse GnRH-R (48), human AR (49), SF-1 (28), P-Otx/Ptx1 (32), H-ras (50), c-myc (51), P-LIM (32), cJun (52), CREB (53), BSK-CAT (42), and Fos (54). Briefly, cDNA clones (1 µg) were spotted in duplicate on two identical Nylon membranes (HybondTM-N+; Amersham Pharmacia Biotech, Cleveland, OH) and hybridized overnight at 65 C in Church’s buffer with either transgenic or nontransgenic probes. Pooled (n = 10–15) total pituitary RNA (20 µg) was used for first-strand cDNA synthesis using Superscript II (Life Technologies, Inc.) in the presence of 60 µCi of [{alpha}-32P]dCTP, nonlabeled dGTP, dATP, dTTP, and random primers (Life Technologies, Inc.). Blots were washed in 2xX SSC, 0.1% SDS (15 min). Data analysis was performed using the Ambis Radioanalytical Imaging System. Each lane on the blot was analyzed separately, and the computer was allowed to identify peaks and determine appropriate background. Net counts from each peak were used in the statistical analysis. To avoid problems with changes in the expression of the housekeeping genes, statistical analysis was performed without normalization of the data. Comparison of transgenic and nontransgenic control was made using a multiway ANOVA. In this analysis, the gene expression response was first transformed using a 1/log(exp) transformation to satisfy normality and variance stabilization assumptions of the analysis. The ANOVA allowed for controlling variation in expression due to different genes, different experiments, and replications within experiments. Interactions of these factors with treatment were also explored. Further comparisons between transgenic and control for each gene were made using specific contrasts derived from the ANOVA. This was done after first examining from the ANOVA whether an overall transgenic effect was present and whether there were any differences in expression between genes. All tests were run at a significance level of P = 0.05 and were two sided.


    ACKNOWLEDGMENTS
 
The authors would like to acknowledge the contributions of the following people whose assistance made this work possible: Dr. M. Susan Smith, Dr. Richard Hanson, Dr. Joe Nadeau, Dr. Kimberly Risma, Dr. Ruth Keri, Rachel Adamek, Kit Sutherland, and David Peck.


    FOOTNOTES
 
Address requests for reprints to: John H. Nilson, Ph.D., Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.

This work was supported by an NIH training grant in metabolism, 5T32DK-07319 (to R.A.), NICHD Grant HD-34032, and NIDDK Grant DK-28559 (to J.H.N.).

Received for publication December 21, 1998. Revision received May 25, 1999. Accepted for publication June 7, 1999.


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