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Ovarian Dependence for Pituitary Tumorigenesis in D2 Dopamine Receptor-Deficient Mice

Vollum Institute, Oregon Health and Science University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Malcolm J. Low, M.D., Ph.D., Vollum Institute L-474, 3181 SW Sam Jackson Park Road, Oregon Health and Science University, Portland, Oregon 97201. E-mail: low{at}ohsu.edu.

	Abstract

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Hypophyseotropic dopamine exerts a tonic inhibitory tone on pituitary lactotrophs by the activation of dopamine D2 receptors (D2R). Ablation of D2R through gene knock-out approaches results in hyperprolactinemia and prolactinomas. This phenotype is more severe and develops more rapidly in female mice. We tested whether the female hypersensitivity is due solely to the loss of D2R inhibitory tone or concomitant stimulation by ovarian factors. C57BL/6J congenic D2R^-/- mice were ovariectomized at 2 months of age and serum PRL levels were measured serially. Ovariectomy attenuated hyperprolactinemia and after 18 months, D2R^-/- mice had average pituitary weights of 4 mg, compared with 60 mg in the intact group. 17ß-Estradiol did not restore PRL secretion or pituitary weight. Although the pharmacologic dose of estradiol slightly increased pituitary weight in wild-type and D2R^-/- mice, it inhibited serum PRL in both intact and ovariectomized females and in castrated males. For comparison, we tested the estradiol response of wild-type 129S6/SvEv mice in the same paradigm and found the expected increase in pituitary weight and serum PRL. Our results demonstrate that the development of hyperprolactinemia and prolactinomas in mice lacking D2R is dependent on ovarian stimulation and likely involves a factor(s) in addition to estrogen. Furthermore, we showed that estradiol-induced proliferation and PRL secretion can be differentially regulated in a strain-specific manner. These findings illustrate the importance of genetic background when analyzing endocrine regulation in mutant mouse models.

	Introduction

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HYPOTHALAMIC DOPAMINE IS a potent inhibitor of lactotroph proliferation and PRL secretion (1). Early studies using hypophysectomy and pituitary implantation under the renal capsule illustrated the importance of hypothalamic factors in regulating pituitary function. Pharmacological and histological approaches led to the understanding that tuberoinfundibular dopamine is responsible for exerting a tonic inhibitory tone on lactotrophs. Subsequently, the dopamine D2 receptor (D2R) was found to be the predominant dopamine receptor subtype in the anterior pituitary (2, 3) and therefore believed to mediate dopamine’s inhibitory actions on lactotrophs. Recently the importance of D2R in maintaining normal lactotroph function has been clearly demonstrated. Mice lacking D2R by homologous recombination display chronic hyperprolactinemia and lactotroph hyperplasia (4, 5). Aged D2R^-/- females (14–18 months) develop macroadenomas and D2R^-/- males (18–21 months) eventually develop microadenomas without concomitant hyperplasia (6). In both male and female D2R^-/- mice, there is a surprisingly long latency to the appearance of lactotroph adenomas. However, the loss of D2Rs causes a much more pronounced effect in the females, including diffuse lactotroph hyperplasia, than the males. The latency to, and the sex-specific progression of, the pathology observed in D2R^-/- mice led us to question whether the loss of dopamine tone is sufficient to cause lactotroph pathology or rather results in a permissive environment for stimulatory factors to function unopposed leading to PRL hypersecretion and lactotroph proliferation.

An interactive balance between dopamine and sex-steroid signaling appears to underlie the regulation of lactotroph function. Estradiol affects PRL secretion directly by acting on the lactotroph to stimulate PRL gene expression and modify lactotrophic responses to other inhibitory and stimulatory factors (7). In addition, estradiol affects lactotroph proliferation and PRL secretion indirectly by altering the activity of hypothalamic neuroendocrine neurons. Direct mitotic and secretory actions of estradiol on lactotrophs have been demonstrated in numerous in vitro and in vivo studies using transplantation, hypophysectomy, and genetic models. Endogenous estrogens regulate lactotroph function in rats and ovariectomy results in decreased PRL release and reduced lactotroph size and numbers (8), effects that can be reversed by the administration of estradiol (9). Immunoneutralization of endogenous estradiol prevents the preovulatory release of PRL during proestrus (10). Furthermore, administration of estradiol to susceptible strains of rat results in the formation of lactotroph adenomas and hyperplasia, which are preceded by a decrease in hypothalamic dopamine production (11).

In addition to direct actions, estrogens also modify lactotrophic responses to physiological inhibitors and stimulators of PRL secretion (7). Acute administration of either the dopamine antagonist sulpiride or estradiol alone to Wistar rats causes a modest increase in lactotroph proliferation, and this mitogenic action is augmented when both agents are given together (12). Administration of an antiestrogen can block the mitotic effect of sulpiride and conversely, estrogen-induced proliferation can be blocked with a dopaminergic agonist (12). Dopamine is a less potent inhibitor of PRL secretion and lactotroph proliferation when estradiol is present (13, 14), further indicating that the balance between estradiol and dopamine is an important factor in the regulation of lactotroph function.

Our previous studies revealed that the loss of dopamine action via D2Rs has a more profound effect on pituitary function in female mice than male mice (4, 6). These data also support the hypothesis that there is a critical relationship between sex steroids and dopamine tone in regulating lactotroph function. However, this relationship is complex as evidenced by the fact that the sex-dependent nature of the pituitary dysregulation in D2R^-/- mice occurs despite similar serum estradiol levels in the D2R^-/- males and females (4, 5). Additionally, basal PRL levels in male and female rats are not highly different, even though hypophyseotropic dopamine in the stalk is 5–7 times higher in females (15). This further illustrates that factors other than estradiol and dopamine are also involved in the regulation of lactotrophic function at the level of both the hypothalamus and the pituitary (7). The expression and actions of many of the growth factors, hypothalamic-releasing factors, and neurotransmitters that affect lactotroph function are themselves regulated by estradiol and/or dopamine. Dopamine expression and release from tuberoinfundibular neurons is regulated largely by PRL (1). Estradiol increases PRL levels and decreases hypophysiotropic dopamine. Therefore, in vivo it has been difficult to determine the relative contribution of decreased dopamine tone resulting from estradiol exposure and direct actions of estradiol and other stimulatory factors on lactotroph proliferation and PRL secretion. In the present study, a D2R-deficient mouse model was used to determine whether exposure to ovarian factors is necessary for the development of pituitary adenomas in the absence of D2Rs.

	Materials and Methods

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Animals
D2R^-/- mice (official strain designation Drd2^tm1low by the Induced Mutant Resource Center at The Jackson Laboratory, Bar Harbor, ME) were generated in our laboratory as previously described (4). All D2R^-/- mice used in these studies were congenic animals generated by backcrossing the mutant allele on the C57BL/6J background for 12 consecutive generations. Wild-type C57BL/6 control mice in these studies were D2R^+/+ siblings from the D2R^+/- matings. Wild-type 129S6/SvEv mice were obtained from Taconic Farms, Inc. (Germantown, NY). All mice were housed in a controlled environment at ambient temperature with a 14-h light/10-h dark cycle. Mouse chow (PMI Feeds Inc., St. Louis, MO) and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Surgery and treatment
At 2 months of age, mice underwent bilateral gonadectomy or sham surgery under isoflurane anesthesia. In some experiments a 17ß-estradiol (Sigma, St. Louis, MO)-filled SILASTIC-brand capsule (Dow Corning Corp., Midland, MI; internal diameter 0.155 cm, length 0.5 cm) or empty control capsule was implanted sc between the scapulae. At the termination of the experiments, mice were killed by decapitation, and pituitaries and uteri were collected.

PRL measurements
Serum was obtained between 1200 and 1400 h under brief isoflurane anesthesia from tail bleeds or trunk blood for terminal measurements and frozen at -80 C until analysis. For analysis of pituitary PRL content, immediately after mice were killed pituitaries were collected, weighed, and homogenized in assay buffer. Homogenates were centrifuged at 1000 g for 30 min at 4 C. A fraction (1–10 µl) of the resulting supernatant was assayed. PRL RIA was performed using mPRL reference preparation AFP6476C, mPRL AFP1077D for iodination, and anti-mPRL antisera AFP131078 (provided by Dr. A. F. Parlow and the National Hormone and Peptide Program) as directed. Mouse PRL was iodinated with 3 µg chloramine-T (Sigma) per microgram peptide, purified with Dowex 20–50 mesh resin beads (Bio-Rad Laboratories, Inc., Hercules, CA), and approximately 20,000 cpm were added to each assay tube. Assay sensitivity was approximately 30 pg/tube. Samples were analyzed in duplicate with all samples from a particular experiment measured in one assay.

Statistical analysis
The data shown in the text and figures are the means ± SEM. Data were analyzed by ANOVA for multiple groups or t test when only two groups were compared. The Newman-Keuls multiple comparison test was used for post hoc analyses.

	Results

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Effect of ovariectomy on serum PRL levels and pituitary weight in D2R^-/- mice
Intact D2R^-/- female mice were hyperprolactinemic starting at 12 wk of age (4) and displayed increasing PRL levels through time (Fig. 1A). The experiment in Fig. 1 was carried out until 18 months after ovariectomy (ovx), and the PRL levels continued to rise in the D2R^-/- mice during this time. In contrast, D2R^-/- females that were ovariectomized did not display increased serum PRL levels over time; the serum PRL did not increase significantly from the initial post-ovx measurement of 200 ng/ml until the 18-month time point. The modest rise in PRL at the older age is similar to that previously observed in male D2R^-/- mice (4, 6). At all time points from 3 months post ovx and beyond, ovariectomized D2R^-/- mice had significantly lower serum PRL than intact D2R^-/- mice.

View larger version (48K): [in this window] [in a new window]   Figure 1. Serum PRL levels and pituitary weights in ovariectomized D2R-/- mice. Mice were ovariectomized at 8 wk of age and followed up for 18 months. A, Serum PRL levels were determined from tail blood collected at 1, 2, 3, 6, 9,12, and 18 months after ovariectomy from ovariectomized D2R-/- mice (hatched bars) and intact control D2R-/- mice (dark bars). Ovariectomy significantly blunted the rise in serum PRL. B, Ovariectomy blocked the increase in pituitary weight caused by the loss of D2Rs. C, The gross appearance of pituitaries from wild-type, D2R-/-, and ovariectomized D2R-/- mice at the termination of the 18-month experiment. Glands from ovariectomized D2R-/- mice were nearly indistinguishable from wild-type glands on gross examination, although there was a slight increase in size. *, P < 0.01, compared with intact D2R-/- control; n = 4–8 mice/group.

Effect of estradiol-17ß on pituitary weight in wild-type and D2R^-/- C57BL/6 mice
Female wild-type mice, whether intact or ovariectomized, displayed increased pituitary weight after an 8-wk exposure to exogenous estradiol (Fig. 2A). The estradiol capsules produced constant pharmacologically elevated serum E2 levels (3.5 ± 1 ng/ml). Ovariectomy significantly decreased pituitary weight in both wild-type and D2R^-/- mice. Estradiol increased pituitary weight in the D2R^-/- ovariectomized females, compared with ovx alone; however, there remained a significant difference between the estradiol-treated ovariectomized D2R^-/- group and the intact D2R^-/- group, suggesting that estradiol-only replacement is not sufficient to maintain the increased proliferative rate in the D2R^-/- ovariectomized mice. Pituitary weights were higher in the intact D2R^-/- females, compared with wild-type females, consistent with our previous findings (4, 6). There was no significant difference in serum estradiol levels between wild-type and D2R^-/- mice (P = 0.441).

View larger version (29K): [in this window] [in a new window]   Figure 2. Pituitary weights in gonadectomized and estradiol-treated mice. Female (A) and male (B) mice either intact (dark bars) or gonadectomized (Gx, white bars) were implanted with empty capsules (nonhatched bars) or 17ß-estradiol-filled capsules (hatched bars). Pituitary glands were collected and weighed after 8 wk of treatment. a, Significant effect of estradiol (P < 0.0001); and b, significant effect of gonadectomy (P < 0.001), compared with matched controls. n for each group is indicated on the bar.

Inhibition of PRL secretion by exogenous estradiol in C57BL/6 mice
Serum PRL levels were determined from the trunk blood obtained at the time of pituitary collection. Ovariectomy decreased PRL levels in wild-type and D2R^-/- mice (Fig. 3A). Surprisingly, estradiol administration also decreased serum PRL levels in intact and ovariectomized wild-type females and in D2R^-/- ovariectomized females. Gonadectomy increased serum PRL in male D2R^-/- mice and estradiol reduced PRL levels in gonadectomized males. D2R^-/- mice of both sexes had higher basal PRL levels than wild-type mice consistent with our previous findings (4). The decrease in serum PRL after estradiol administration reflects a deficit in secretory release because pituitary PRL contents were increased by estradiol in the same mice (Fig. 3C).

View larger version (26K): [in this window] [in a new window]   Figure 3. PRL levels in gonadectomized and estradiol-treated mice. Female wild-type and D2R-/- (A) and D2R-/- male (B) mice either intact (dark bars) or gonadectomized (white bars) were implanted with empty capsules (nonhatched bars) or 17ß-estradiol-filled capsules (hatched bars). Trunk blood was collected after 8 wk of treatment, and serum PRL levels were determined. a, Significant decrease in PRL after estradiol (P < 0.001); and b, and significant response to gonadectomy, compared with intact control (P < 0.001). Pituitary PRL content was determined (C) in gonadectomized male D2R-/- mice implanted with an empty capsule (nonhatched bar) or a 17ß-estradiol-filled capsule (hatched bar) for 8 wk. Estradiol caused a significant increase in pituitary PRL content (a, P = < 0.001). n for each group is indicated on the bar.

View larger version (29K): [in this window] [in a new window]   Figure 4. Pituitary weights and serum PRL levels in estradiol-treated 129S6/SvEv mice. Intact female 129S6/SvEv mice were implanted with estradiol-filled (hatched bars) or empty capsules (nonhatched bars) for 8 wk. Pituitary weights (A) and serum PRL levels (B) were determined. *, P < 0.001; n = 6 mice/group.

View larger version (30K): [in this window] [in a new window]   Figure 5. Uterine enlargement following estradiol treatment in both strains of mice. Following the 8-wk treatment period, uteri were collected and examined. A, In both 129S6/SvEv (left side of panel) and C57BL/6 (right side of panel) mice, there was a noticeable increase in uterine size after estradiol exposure (E2), compared with control (C) conditions. B, The graph indicates the uterine weights at the end of the treatment (C, open bars; E2, solid bars). *, P < 0.01. n = 3–5 mice/group. There was also a smaller but significant difference in uterine weight between the control mice from each genotype (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

Ovariectomy results in decreased serum PRL levels in rats and mice. This is caused, at least in part, by the loss of estrogen action at both the levels of the pituitary and the hypothalamus. Ovariectomy suppresses tuberoinfundibular dopamine (TIDA) neuron activity and this can be reversed by estradiol treatment (9). Interestingly, endogenous estradiol appears to increase the activity of TIDA neurons because there is higher basal activity in females (18, 19). However, in vitro experiments have revealed that estradiol can directly decrease TIDA neuron activity (20). Estradiol also increases serum PRL levels and lactotroph proliferation via direct actions on lactotrophs including the down-regulation of D2R (13). TIDA neurons are responsive to acute and chronic changes in serum PRL except during pregnancy and lactation or in mice with prolactinomas when the neurons become refractory to PRL so that high PRL levels can be maintained. Under normal conditions, PRL increases TIDA dopamine production and/or release via short-loop feedback to inhibit further PRL release. In the D2R^-/- mouse model, this level of regulation is still intact (20A ), but the signaling mechanism for the hypophyseotropic dopamine is nonfunctional. Here we have shown that it is not directly the lack of dopamine signaling that causes lactotroph proliferation and hyperprolactinemia but rather the unopposed stimulation from ovarian factors.

Differential roles of estradiol
We hypothesized that the reduction in pituitary growth and PRL secretion of D2R^-/- mice after ovariectomy could be accounted for by the resulting decrease in estradiol levels. To test this possibility, we treated ovariectomized D2R^-/- mice with exogenous estradiol for up to 2 months. Surprisingly, estradiol did not increase serum PRL levels but did modestly increase pituitary weight. This finding is consistent with findings by Sinha and Gilligan (21), who demonstrated that high doses of estradiol did not increase basal or disinhibited concentrations of serum PRL despite considerable increases in pituitary weights. These authors proposed that high doses of estradiol may modify dopaminergic receptors in the pituitary gland and/or brain in a manner to account for the lack of stimulation by estradiol or dopamine receptor antagonists. If this were true, one would expect that estradiol would have less of an effect in the D2R^-/- mice, compared with wild-type mice; however, we found very similar responses in both wild-type and D2R^-/- mice. Alternatively, high doses of estradiol may alter the expression or actions of other pituitary and hypothalamic factors that regulate PRL secretion.

Although estradiol did not increase circulating PRL levels, it did increase pituitary PRL content in both male and female mice indicating that, at least at superphysiological doses, estradiol can specifically interfere with PRL release. This is consistent with previous findings in S/W mice (21), although the mechanism by which estradiol inhibits release is not yet clear. Our data comparing D2R^-/- and wild-type mice demonstrate that it is likely not a dopamine-mediated effect. One possibility is that estradiol may interfere with the actions of hypothalamic-releasing factors leading to storage of PRL within the lactotrophs. It is also feasible that estradiol may alter proteolytic cleavage or other posttranslational modifications of PRL that could inhibit its secretion, although this has not yet been studied in detail in vivo.

It is important to note that in the present studies, estradiol was administered at pharmacological doses in a static manner. We cannot rule out the possibility that either physiological estradiol levels or estradiol administered at varying doses to mimic the estrous cycle may be able to reverse the effects of ovariectomy in D2R^-/- mice. It is possible that PRL secretion is tightly regulated by estrogen levels or fluctuations, whereas proliferation and pituitary PRL content may be less sensitive to these parameters. One study (22) has indicated that in a cell line, estradiol can differentially regulate PRL release and lactotroph proliferation depending on the dose. However, if PRL release is specifically inhibited by static elevations in estradiol, it is inhibited in a strain-specific manner because estradiol administered under the same conditions in 129S6 mice did cause an increase in serum PRL levels.

Genetic contribution to estradiol actions
To determine whether the inhibitory effect of estradiol on PRL secretion was a specific action of estradiol or rather a strain-specific pharmacological effect because of the high dose, we evaluated the lactotroph response to estradiol in another strain of mice. Using the same amount of estradiol and length of exposure, we found that estradiol increased both serum PRL levels and pituitary weights in 129S6/SvEv mice, consistent with a published report by Wynick et al. (17) indicating the strain-specific nature of estradiol responses. There are many discrepancies as to the nature and extent of estrogen’s action in several animal models and in humans. In humans PRL levels coincide with hormonal status and lactotrophs proliferate before lactation to maintain elevated PRL levels. However, exogenous estradiol administration in humans has little effect on lactotroph function except in highly susceptible persons and when very high doses are administered (1, 23). In contrast, exogenous estradiol is a potent stimulator of PRL secretion and lactotroph proliferation in certain strains of rodents. Fischer 344 rats are the most widely studied and most sensitive rat strain to estrogen-induced pituitary growth. Other strains are also sensitive to estradiol including ACI, Wistar-Furth, and Copenhagen (24, 25, 26, 27), whereas some strains are relatively insensitive to estrogen-induced pituitary tumorigenesis including Brown-Norway, Holtzman, and Sprague Dawley (28, 29, 30).

In addition to differences in estradiol sensitivity among strains of rat, there are likely varied responses between rats and mice. Caution must be used when comparing data from these two species. However, genetic regulation of estrogen-induced pituitary growth has been less well characterized in mice than rats. In one study, Gardner and Strong (31) presented a comparison of pituitary responses in seven different mouse strains (A, C³H, CBA, C12I, JK, N, and C57). These authors found that only C57 mice displayed significant hyperplasia (15 of 106 mice) after approximately 250–450 d of estrogen treatment and that higher doses of estradiol did not increase the initiation or severity of tumorigenesis, although this was difficult to interpret because high doses were associated with earlier mortality from nonpituitary causes. More recently other investigators have reported that C57 mice are relatively refractory to estradiol-induced pituitary tumorigenesis (32). Some of the discrepancies in the literature may be due to classifications of hyperplasia vs. tumor, the dose, chemical form, and duration of estrogen treatment and to the use of various substrains of mice. In our study, we found that estradiol exerted strain-specific effects on PRL release but that both 129S6 and C57BL/6 mice displayed comparable increases in pituitary weight. The estradiol response that we observed in C57BL/6 mice is similar to that which Sinha and Gilligan reported in their study using S/W mice (21).

Genetic variations in nonpituitary responses to estradiol have also been reported in mice (33, 34, 35). Tissue-specific strain differences were seen in our studies as well. The lack of estrogen-induced PRL release that we observed in C57BL/6 mice prompted us to determine whether this strain may be refractory to exogenous estradiol in general. We found that estradiol caused the expected increase in uterine morphology in both C57BL/6 and 129S6 mice, consistent with previous findings in rat. Despite the increased pituitary sensitivity to estradiol in Fischer 344 rats, uterine responses are comparable to those in pituitary-insensitive strains (30). Furthermore, other investigators have demonstrated that a 1-wk treatment of C57BL/6 mice with estradiol induced pituitary galanin gene expression in an estrogen receptor -dependent manner (36).

Mechanisms underlying strain-specific responses to estradiol remain largely unknown. Several studies have recently been undertaken to map genetic loci that may be responsible for estrogen sensitivity in various tissues, and a few loci have been identified (27, 29, 33). Anatomical differences that could potentially mediate differences in tumorigenesis and endocrine responses between strains have also been detected in rats and mice. Of particular interest to the data presented here is the observation by other investigators that there are notable strain differences in the distribution of hypothalamic galanin neurons. There is a small tight cluster of galanin immunoreactive cells adjacent to the third ventricle and ventral to the preoptic area galanin neurons in 129 mice that is absent in C57BL/6 mice (37). This cluster is also present in DBA/2J mice (38) but not in CD-1 mice (37), raising the possibility that strain-specific variations in galanin immunoreactivity may be common in mice.

Galanin expression and serum galanin levels are highly dependent on estrogen status. Mice of the 129P2 genetic background lacking galanin via disruption of the gene display diminished estrogen-induced PRL release, compared with wild-type controls (17). It is an intriguing possibility that galanin produced from the cell cluster that is absent in the C57 mice is necessary to mediate estrogen-induced PRL release; however, this has not yet been studied. Strain-specific differences in pituitary galanin expression appear less likely as an explanation for the varying responses to PRL secretion because, as noted earlier, estradiol increased galanin gene expression in the pituitaries of C57BL/6 mice (36). However, the estrogen-sensitive nature of galanin-induced lactotroph proliferation at the level of the pituitary (39) suggests the possibility that there could be subtle differences in estradiol/galanin interactions among strains that could mediate differential responses over time. Galanin is just one potential peptide that could mediate strain-specific responses to estradiol. Expression patterns of the estrogen receptor subtypes and specific dynamics of estrogen receptors likely also mediate tissue and strain differences to estradiol responses. There is a myriad of other peptides, genes, and anatomical features that could underlie strain-specific responses to estradiol as well as responses to a variety of hormones, peptides, and pharmacological agents. The data presented here and many other recent studies illustrate the need to consider strain differences when using mice for neuroendocrine investigations. Our data illustrate that there are not only differences in sensitivity to estradiol between strains but that different strains can display opposing responses in pituitary PRL secretion to estradiol.

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
In the present study, we have shown that hyperprolactinemia and hyperplasia in D2R^-/- mice are dependent on ovarian stimulation. This is the first in vivo study to clearly demonstrate that escape from dopamine inhibition alone is not sufficient to cause severe lactotroph dysregulation, but rather pituitary growth is dependent on stimulation by an ovarian factor. The fact that estradiol treatment did not reverse the ovariectomy-induced decrease in serum PRL reflects a strain-specific response to superphysiological doses of estradiol. The inability of estradiol to increase serum PRL levels in C57BL/6 mice is independent of D2R mediation because responses were equivalent in D2R^-/- and wild-type mice. Ovariectomy abrogated the lactotroph dysfunction caused by D2R inactivation, but chronic estradiol did not reverse this change, indicating that either another ovarian factor mediates the stimulatory response or that exposure to physiological patterns of estradiol is more effective in promoting hyperplasia/hyperprolacinemia in lactotrophs of C57BL/6 mice than constant exposure to high concentrations of estradiol.

	Acknowledgments

 
We thank Dr. D. Hess and the RIA core lab at the Oregon Regional Primate Research Center for the measurements of estradiol and J. Notis for assistance with the photography.

	Footnotes

 
This work was supported by NIH Grant T32-DK-07674 (to S.T.H.).

Abbreviations: D2R, D2 receptor; ovx, ovariectomy; TIDA, tuberoinfundibular dopamine.

Received April 19, 2002.

Accepted for publication August 15, 2002.

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