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Molecular Endocrinology, doi:10.1210/me.2005-0195
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Molecular Endocrinology 20 (1): 204-211
Copyright © 2006 by The Endocrine Society

Hypothalamic 3',5'-Cyclic Adenosine Monophosphate Response Element-Binding Protein Loss Causes Anterior Pituitary Hypoplasia and Dwarfism in Mice

Theo Mantamadiotis, Oliver Kretz, Stephanie Ridder, Susanne C. Bleckmann, Dagmar Bock, Hermann-Josef Gröne, Jordane Malaterre, Sebastian Dworkin, Robert G. Ramsay and Günther Schütz

Molecular Biology of the Cell I (T.M., S.R., O.K., S.C.B., D.C., G.S.), Department of Cellular and Molecular Pathology (H.J.G), Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany; Institute of Anatomy and Cell Biology, University of Freiburg (O.K.), 79104, Freiburg, Germany; and Peter MacCallum Cancer Centre (T.M., J.M., S.D., R.G.R), East Melbourne 3002, Australia

Address all correspondence and requests for reprints to: Theo Mantamadiotis, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, 8006 Victoria, Australia. E-mail: theo.mantamadiotis{at}petermac.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The principal regulation of body growth is via a cascade of hormone signals emanating from the hypothalamus, by release of GHRH, which then directs the somatotroph cells of the pituitary to release GH into the blood stream. This in turn leads to activation of signal transducer and activator of transcription 5-dependent expression of genes such as IGF-I in hepatocytes, acid labile substance, and serine protease inhibitor 2.1, resulting in body growth. Here, using conditional cAMP response element binding protein (CREB) mutant mice, we show that loss of the CREB transcription factor in the brain, but not the pituitary, results in reduced postnatal growth consistent with dwarfism caused by GH deficiency. We demonstrate that although there appears to be no significant impact upon the expression of GHRH mRNA in CREB mutant mice, the amount of GHRH peptide is reduced. These findings show that CREB is required for the efficient production of GHRH in hypothalamus, in addition to its previously reported role in pituitary GH production and somatotroph expansion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HORMONAL HIERARCHY for regulating the control of body size is complex, requiring the coordinated production of stimulatory (GHRH) and inhibitory (somatostatin) factors in the hypothalamus. Transcriptional and translational controls, as well as complex posttranslational processes, achieve coordination of growth signals at the cellular level. Aside from the direct GH stimulatory effects of GHRH on the somatotrophs of the anterior pituitary, GHRH also promotes somatotroph proliferation (1, 2).

Both in vivo and in vitro studies have demonstrated that a number of transcription factors regulate GHRH and GH expression (3, 4, 5). One of the first transcription factors shown to have an impact upon somatic growth was cAMP response element (CRE)-binding protein (CREB). CREB and the related proteins CRE-modulatory protein (CREM) and activating transcription factor 1 (ATF1) form a subfamily of leucine zipper transcription factors. These proteins are activated by phosphorylation and bind as homo- or heterodimers to the CRE of the promoters of their target genes (6). The role of CREB in growth regulation was shown by expression of a dominant-negative CREB in the anterior pituitary. These mice displayed severe anterior pituitary hypoplasia caused by somatotroph loss and, as a consequence, exhibited low GH levels and dwarfism.

Generation of mice with either total or tissue-specific loss of CREB has revealed the importance of this transcription factor in normal physiology. Complete loss of CREB results in early postnatal death due to failure of normal lung development (7), whereas loss of CREB in thymus results in a proliferative defect of T cells (8). Loss of CREB in vivo consistently results in up-regulation of CREM, which is a compensatory cellular survival response (9, 10). The functional compensation by CREM in nerve cells has been demonstrated in mice where brain-specific CREB loss was achieved on a total CREM–/– background (11). In these mice, perinatal death occurred due to late embryonic neuronal death and the inability of newborn mutant mice to suckle. During the generation of the Creb1-Crem compound mutant mice, we noticed that mice that had lost only CREB in brain were indistinguishable from littermates at birth but were invariably smaller than littermates after a few days, indicating that CREM was able to compensate for neuronal loss but not for somatic growth.

Through the use of the conditional CREB knockout mice and the nestin-Cre-recombinase transgenics (12), CREB was specifically deleted in brain but not in the pituitary. The findings presented in this study show that although CREB was not lost in the pituitary, there was a reduction in GH levels. CREB therefore functions at two levels within the hypothalamic-pituitary growth axis, namely the hypothalamus and, as previously shown, the pituitary (6).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CREB Loss in Brain But Not Pituitary
Previous studies have shown that the rat nestin promoter and enhancer allow transgenes to be expressed at the earliest phases of central nervous system development (13, 14). In the study presented here, CREB loss, as detected by immunohistochemical analysis (IHC) was evident in the neuroepithelium from embryonic d 12.5 (E12.5) (data not shown). The specificity of nestinCre-mediated Creb1 recombination at birth was determined by IHC detection of CREB. Loss of CREB and mRNA in brain including the hypothalamus of mutant mice, but not in pituitary cells, was seen (Fig. 1Go, A–D and I). Wild-type (Fig. 1EGo) CREB expression was maintained in the mutant adult mouse anterior pituitary lobes (Fig. 1FGo). The related CREB family members, CREM and ATF-1, were also expressed at normal levels in the pituitary gland of both control (Fig. 1Go, G and H) and mutants (data not shown).



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  		Fig. 1. CREB1NesCre Mutant Mice Have a Loss of CREB in Brain But Not the Pituitary Gland

At E18.5 (A–D) CREB is expressed throughout the brain in Creb1loxP/loxP mice (A) but lost in CREB1NesCre brains (B). The specificity of CREB loss is highlighted by the presence of CREB in the pituitary but not in the rest of the brain, including the hypothalamic regions (A–D). In the adult anterior pituitary lobe, CREB expression persists (E and F) in both Creb1loxP/loxP and CREB1NesCre mice. This is also highlighted by the specific loss of CREB mRNA signal in the anterior pituitary but not in the hypothalamus (I). The CREB-related factors CREM and ATF-1 are also expressed in the pituitary glands of wild-type mice (G and H). ant, Anterior; int, intermediate; pos, posterior; pit, pituitary; hyp, hypothalamus.

 
Mice Lacking CREB in Brain Are Dwarf and Exhibit Pituitary Hypoplasia
By disrupting the Creb1 gene in the germline, we previously showed that loss of CREB results in rapid postnatal death due to a developmental delay and failure to breathe normally (7). When both CREB and CREM were lost in brain, neuronal death occurred (11). Although CREM was able to compensate for CREB loss with respect to neuronal survival, CREB clearly has specific functions that cannot be attributed to other related factors. Here we report that although there was no difference observed between mutant and control mice at birth, by 7 d CREB1NesCre mice were invariably smaller (P < 0.05) than control littermates, as indicated by the growth curves (Fig. 2Go, A and B). Although there are differences in the GH/IGF-I axis of female and male mice, in CREB1NesCre mice, both males and females were affected, and on average reached only 70–80% of control adult weight (Fig. 2BGo). At E18.5, just before birth, the pituitary glands of CREB1NesCre mice were indistinguishable from control mice (Fig. 1Go, C and D). However, the pituitary glands of adult CREB1NesCre mice were smaller than in control littermates (Fig. 3Go).



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  		Fig. 2. Dwarfism in CREB-Deficient Mice

A, Body size differences were apparent after 7 d of age and persisted throughout the life of both male and female mice. female mice (10 wk old) are shown here. B, Comparison between CREB1NesCre (mutant) and control mice are shown up until 80 d of age. The weights are calculated from data gathered from both male (n = 4) and female (n = 2) mice and are significantly different from d 7 onward (*, P < 0.05 by paired t test). The inset shows the weight gain/growth rate of individual male and female mutant and control mice. c, Control; m, mutant.

 


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  		Fig. 3. Anterior Pituitary Hypoplasia and Reduced Pituitary GH and Liver IGF-I Levels in Female CREB-Deficient Mice

CREB1NesCre mice displayed severe hypoplasia of the anterior pituitary lobes (A, C, and E) compared with control pituitary glands (B, D, and F). There are fewer GH expressing somatotroph cells in the anterior pituitary of CREB1NesCre mice (G) compared with controls (H). Insets to panels G and H show that, as expected, there is no GH in the posterior lobes. Semiquantitative RT-PCR and densitometric analysis showed that liver Igf-1 mRNA levels were reduced 3.6-fold (n = 2; P < 0.0005) in CREB1NesCre mutant mice (m) compared with controls (c) in panel I. Scale bars, 1 mm.

 
Analysis of Pituitary Hormones and IGF-I Expression
To assess pituitary hormone levels, serum pooled from at least three 8-wk-old mice was measured using RIA. Analysis of various pituitary hormones was consistent with a GH deficiency (Table 1Go). GH levels were severely reduced when examined by both IHC (Fig. 3Go, G and H) and RIA (Table 1Go). Serum GH levels in CREB1NesCre mice were less than 0.16 ng/ml, compared with 3.2 ng/ml in control mice. Consistent with a GH deficiency was a 3.6-fold (n = 2, P < 0.0005) reduction in IGF-I mRNA expression in the liver (Fig. 3IGo). Prolactin (PRL) levels were similarly reduced in CREB1NesCre mice, less than 0.08 ng/ml compared with 1.6 ng/ml in control mice. FSH levels were 12.4 ng/ml in CREB1NesCre mice, compared with 40 ng/ml in control mice. LH levels were near the limits of detection with no demonstrable difference in LH between CREB1NesCre and control mice, although IHC showed that the number of LH-positive cells were roughly 35% of control LH-positive cell numbers (Fig. 4Go). TSH (4 ng/ml) and T4 (20 ng/ml) levels were similar in CREB1NesCre and control mice. To extend these data, we performed IHC on pituitaries of mutant and control pregnant female mice (Fig. 4Go). Because the pituitaries of mutant mice were invariably smaller than controls, we counted the number of PRL-, FSH-, and LH-positive cells in the representative matched sections shown in Fig. 4Go. The data derived from these measurements showed that PRL-, FSH-, and LH-positive cells in mutant pituitaries were 22%, 46%, and 35% of control numbers, respectively.


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  		Table 1. Serum Levels of Pituitary Hormones (ng/ml) in Adult Female Mice

 


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  		Fig. 4. Failure of Alveolar Expansion in Postpartum CREB1NesCre Mice

Whole-mount staining of mammary glands with iron-hematoxylin reveals the thinly branched mammary ducts and alveoli in postpartum CREB1NesCre mice, compared with the milk-filled ducts and alveoli in control postpartum females. Overall expression of PRL, FSH, and LH in the pituitaries of pregnant female mice was lower in CREB1NesCre mice, compared with control pregnant female mice. Pituitaries: controls, x1.2 magnification; mutant, x2 magnification. Mam, Mammary glands.

 
Lactation and Fertility Defect
When 10-wk-old CREB1NesCre female mice were crossed with wild-type C57BL/6 mice, only 20% (four of 20) of mutant females became pregnant and gave birth to live pups over an 8-wk breeding period, compared with 80% (16 of 20) of wild-type C57BL/6 females. Newborn pups irrespective of genotype, born to CREB1NesCre mothers did not survive beyond the first day due to a lactation failure of the mother. The pups born to CREB1NesCre mothers showed signs of dehydration and lack of visible milk deposit in their stomachs. To determine mammary gland expansion in the CREB1NesCre mothers, whole-mount mammary glands were prepared to examine alveolar structures. In lactating control mice the alveoli were densely stained, indicative of efficient mammary gland expansion and lactation, whereas CREB1NesCre mammary structure showed less dense alveoli, indicative of poor expansion and lactation (Fig. 4Go).

When mice were mated with wild-type C57BL/6 females, four males from 12 crosses resulted in the birth of live litters, compared with nine litters from 12 crosses from wild-type C57BL/6 mice. Approximately 80% of sexually mature males showed either unilateral or bilateral cryptorchidism, which may be caused by the reduced GH levels (15). Examination of testicular weight showed that the average testicular weight of mice (68 mg ± 35 mg per testis; n = 4) was less than half that of control (157 mg ± 23 mg per testis; n =4) mice. When the smaller body weight of mice was accounted for, the ratio of the calculated testis weight index for mutant vs. control mice was significantly smaller (0.74; P = 0.035, Student’s t test), indicating that, even accounting for body size, mutant testes were smaller. Sperm counts were not significantly different in mutant mice compared with controls; although sperm motility appeared to be impaired, this was not assessed further.

Expression of Hypothalamic Factors
Because the loss of CREB was restricted to the nervous system, we examined a number of hypothalamic factors, which can stimulate or inhibit pituitary hormone synthesis and release. Examination of hypothalamic mRNA for GHRH and somatostatin by RT-PCR (CREB1NesCre, n = 6; control, n = 6), RNase protection assay (CREB1NesCre, n = 3/n = 2; control, n = 3/n = 2) and in situ hybridization (CREB1NesCre, n = 2; control, n = 2) showed no differences in expression between CREB1NesCre and control mice (Fig. 5Go). Because hypothalamic hormones reach the pituitary via the median eminence (ME), which is the primary capillary network of the hypophysial portal system and where these factors are most concentrated, we used an antimouse GHRH antibody (16, 17) to perform IHC on brain sections containing the MEs, of young mice (20–24 d old, n =4), an age when GHRH levels are high. There was a clear and significant reduction in GHRH signal in the ME (Fig. 6Go) of CREB1NesCre (n = 4) compared with control mice (n = 4). Image analysis-densitometry showed mutant ME expression at 31% of control ME (n = 4 each genotype; P < 0.003). These data suggest that CREB has a role in regulating GHRH posttranscriptionally. GHRH posttranscriptional processing involves a complex series of enzymatic steps to generate the bioactive peptide (18), and CREB may influence the expression of one or more factors involved in these processes. Whether the same mechanism is responsible for deficient production of PRL and FSH is not known. Further analysis of brain sections by IHC showed no apparent differences in ME levels of somatostatin and GnRH (Fig. 6Go).



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  		Fig. 5. The Expression of Ghrh and Somatostatin (Som) mRNA Is Not Perturbed in Female CREB1NesCre Mice

Semiquantitative RT-PCR (A), RNase protection assay (B) using total hypothalamic RNA as template and in situ hybridization (C) of the hypothalamus showed no reduction in Ghrh and Som mRNA levels of 3-wk-old CREB1NesCre mutant (m) and control mice (c). Representative data are shown. Som, Somatostatin; Cyclo, cyclophilin.

 


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  		Fig. 6. Reduced GHRH Protein Expression in the ME

Female mice (3 wk old) showed reduced GHRH protein expression in the median eminence, which is the primary capillary network of the hypophysial portal system. Stained sections from two different mutant and control mice are shown (four animals of each genotype were analyzed with the same result). Densitometric analysis comparing control and mutant GHRH IHC signals in the MEs (n = 4 each genotype), revealed a 69% reduction over GHRH signal in control MEs (n = 4 each genotype; P < 0.003). Consistent with mRNA signals, somatostatin (Som) and GnRH protein levels were not changed in mutant mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Correct anterior pituitary development requires the specification and appearance of five distinct cell types, characterized by their ability to synthesize and release trophic hormones in response to specific hypothalamic signals. Whereas pituitary-specific transcription factors regulating pituitary development and function have been well characterized (3, 4, 5), transcription factors involved in hypothalamic growth regulation are less well studied. Interestingly, CREB, an important transcription factor with many functions in the central nervous system (11, 19, 20, 21), has been shown to be required for efficient pituitary somatotroph proliferation and function in vitro and in vivo. CREB is one of the major transcription factors activated when GHRH binds to its receptor on somatotrophs. This resulting expression of downstream CREB target genes in turn regulates somatotroph growth and differentiation (22). When a transcriptionally defective dominant-negative CREB was overexpressed under the control of a rat GH promoter, the resulting transgenic mice showed severe anterior pituitary hypoplasia and somatotroph loss (6). By using a conditional CREB1 knockout mouse to direct CREB loss to the brain, but not pituitary gland, we observed a phenotype remarkably similar to the pituitary-specific dominant-negative CREB transgenic mouse (6).

Although CREB1NesCre mice were indistinguishable from control littermates at birth, by 7–10 d of age they were invariably smaller, and both males and females remained proportionally smaller. Analysis of the pituitary glands of adult CREB1NesCre mice showed that the anterior pituitary lobes were severely hypoplastic, similar to that observed in mice overexpressing a dominant-negative CREB in somatotrophs (6). In our model, pituitary CREB expression was normal; thus the pituitary hypoplasia is likely due to hypothalamic CREB loss. Furthermore hypothalamic GHRH, a hormone that promotes somatotroph proliferation, was decreased in mutant mice. Analysis of pituitary hormones showed that GH, PRL, FSH, and LH were reduced in CREB1NesCre mice (Table 1Go and Fig. 4Go). GH deficiency would account for the reduced somatic size and reduced IGF-I mRNA levels in liver (Fig. 3IGo). The overall phenotype of CREB1NesCre mice appears to most closely resemble the little (lit) mutant mice, where GH and PRL are low and there is subfertility but no evidence of hypothyroidism (23). The CREB1NesCre mice also share characteristics of other growth-retarded mutant mice described previously (5), including GH-deficient Ames and Snell dwarf mice (24, 25, 26).

Although GHRH protein expression was reduced in the ME of CREB1NesCre mice, no differences in hypothalamic Ghrh mRNA expression between CREB1NesCre and control mice were apparent. This implies that CREB loss in the hypothalamus does not lead to a detectable difference in Ghrh mRNA expression. In accordance with this observation is the absence of canonical cAMP response elements (CREB binding sites), up to 1200 nucleotides upstream of the transcription start site of the Ghrh gene promoter [analyzed using MAPPER online analysis (27)]. This observation leads to the conclusion that CREB does not regulate GHRH transcription directly, but is rather involved in the regulation of one or more steps in the complex posttranscriptional processing of the pre-pro-GHRH polypeptide. Analysis of some candidate factors, proteolytic converting enzymes PC1/3 and PC2 (28, 29) and secretogranin II, which may be involved in CREB-mediated GHRH processing and release showed no change in mRNA expression of these genes (data not shown). Analysis showed no apparent differences in ME levels of somatostatin and GnRH, although the promoters of these genes harbor CREB binding sites (CREs). Given that multiple transcription factors within transcriptional networks regulate genes and because we show that CREB loss does not measurably affect somatostatin and GnRH expression, the binding and activity of other transcription factors to these promoters are presumably sufficient to maintain normal expression levels.

Apart from the attenuated GH axis, CREB loss also caused a reduction in the fertility of both male and females. When CREB1NesCre females did become pregnant and gave birth, their capacity for lactation was severely impaired, as indicated by the failure of alveolar expansion. Low FSH could explain the reduced fertility, low PRL, and the failure to lactate normally. As GnRH levels appeared to be unaltered in female CREB1NesCre mice, the reduction of FSH and PRL could be caused by the reduced GH and IGF-I levels, which can regulate the hypothalamic-pituitary-gonadal axis (reviewed in Ref.30). Indeed, IGF-I can directly exert an effect upon ovarian and testicular function to also alter LH and FSH expression and release (30, 31). It is interesting to note that CREB1-Ser142 point-mutant mice have defects in circadian rhythm (32), consistent with the proposed role of CREB in the suprachiasmatic nucleus, possibly altering the pulsatile nature of hormone release. Although we did not assess the biological clock of CREB1NesCre mice, the CREB1-Ser142 mice have no defect in fertility or in body size (32).

In summary, the outcome of the study described herein was that brain-specific CREB loss resulted in a phenotype similar to the pituitary-specific dominant-negative CREB transgenic mouse (6) albeit by a different mechanism. This shows that CREB is a key transcription factor, regulating vertebrate somatic growth at multiple levels of the hypothalamic-pituitary-somatomedin axis, including both the hypothalamus and pituitary. Whether mutations of the Creb1 gene account for a subset of syndromes affecting human somatic growth remains to be seen. This is of interest because in the hypothalamus, at least, the related family members, CREM and ATF-1, do not compensate for loss of CREB. Therefore, CREB function in the hypothalamus is essential for normal somatic growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of Mutant Mice
Creb1loxP were generated as described previously (11) and were crossed to nestinCre transgenic mice (12) to generate CREB1NesCre, with Creb1lox/lox mice serving as controls. Two successive crosses were necessary to obtain mutant mice homozygous for the Creb1loxP allele and harboring one allele of the nestinCre transgene, with the second cross being Creb1WT/loxP-nestinCre x Creb1loxP/loxP mice. In this case, mutant mice were obtained at the expected Mendelian ratio of 25%. Mice were bred in a genetic background comprising of a mix of 129SvOla and C57BL/6. Genotyping of mice with the Creb1loxP allele was performed using PCR primers as previously described (33), whereas the nestinCre transgene was detected by tail DNA slot blot analysis. Control animals in all experiments were Creb1loxP/loxP without the nestinCre transgene. Mice used in this study were generally between 2 and 10 wk old. All mice were maintained in accordance with the institutional (Deutsches Krebsforschungszentrum DKFZ and Peter MacCallum Cancer Centre), federal (Federal Republic of Germany and Australia) and state laws governing animal handling and use.

Immunohistochemical Analysis
Mice were perfused with cold 4% paraformaldehyde, and brains were dissected and postfixed for 16 h in paraformaldehyde at 4 C before embedding in paraffin. Paraffin sections were sectioned on a microtome at a thickness of 7 µm. CREB, CREM, and ATF-1 antibodies were used at dilutions of between 1:3,000 and 1:10,000 as described previously (11, 34). Antibodies recognizing rat and mouse GH, FSH, TSH, LH were used at various dilution. Dr. Nobuhiro Miki (Tokyo Women’s University Hospital, Tokyo, Japan), kindly provided the antimouse GHRH antibody. Sections were immunostained using the ABC-Vectastain kit according to the manufacturer’s instructions.

Hormone Measurements by RIA
Serum was collected from 8-wk-old mice and assayed for pituitary hormones as recommended by the manufacturer’s instructions (ICN Diagnostics, West End, NC). Serum from three mice from both CREB1NesCre and control littermates was pooled and used per measurement.

Isolation and Detection of mRNA
Total RNA was isolated using Trizol (Roche, Indianapolis, IN) from a 5-mm3 portion of liver. RT-PCR analysis was performed by using 1 µg RNA to prepare cDNA and amplification by using the following: for detection of Creb1, primers 5'-gctggctaacaatggtacgga-3' and 5'-tccctgttcttcattagacgg-3'; for Igf-1, primers 5'-ctgacctgctgtgtaaacgac-3' and 5'-gtgtggcgctgggcacggata-3'; for Ghrh, primers 5'-atgctgctctgggtgctctttgt-3' and 5'-gagctgaagcagaagtaacagg-3'; and for Som, primers 5'-ctgcctgaggacctgcgacta-3' and 5'-ccattgctgggttcgaattgg-3'. To detect the housekeeping ß-actin, forward 5'-gtgggccgctctaggcaccaa-3' and reverse 5'-ctctttgatgtcacgcacgatttc-3' primers were used. The amplification conditions used were 94 C, 4 min followed by 25 cycles (Igf-1; Ghrh; Som) or 22 cycles (ß-actin) of 94 C, 30 sec; 55 C, 30 sec; 72 C, 40 sec.

RNase protection assays were performed using 10 µg hypothalamic total RNA and was combined with 1 µl [{alpha}32-P]UTP-labeled mouse Ghrh probe, encompassing a 180-bp cDNA fragment or a 5'-somatostatin fragment (from Image clone p998D021138) encompassing 200 bp. The hybridization (56 C overnight) and RNase digestions were performed using the Ambion RPA III kit reagents (Ambion, Inc., Austin, TX). Yeast RNA was included as a negative control. Protected products were resolved on a 5% acrylamide/8 M urea/1xTBE gel. A Cyclophillin probe was used as an internal loading control.

In situ hybridizations were performed on paraffin sections as described in Ref.35 . A 362-bp Ghrh probe encompassing the complete open reading frame and a 521-bp Somatostatin probe harbored in IMAGE clone p998D021138 were used. Sense probes served as negative controls.

Whole-Mount Mammary Gland Staining
Mammary glands from postpartum female mice were dissected and spread onto glass slides and allowed to air dry. The tissue was fixed in Tellyesniczky’s fixative [70% ethanol-formaldehyde-glacial acetic acid (20:1:1)] for 24 h. The following day the fixed tissues on slides were rinsed in running tap water for 1 h. Immersion in three changes of acetone over 2 d was performed to defat the tissue. Hydration of the samples was then done through an ethanol series of 100%, 95%, and 70% for 1 h each. Tissues were stained in freshly made iron-hematoxylin (80 mg FeCl2·H2O for 1.5 h; 14.5 ml 1 M HCl; 87 ml 100% ethanol; 100 mg hematoxylin per 112 ml in water). Tissue was then dehydrated through an ethanol series of 50%, 70%, 95%, and 100% ethanol, followed by two changes of toluene over 2 h.

Imaging, Morphometric, Densitometric, and Statistical Analysis
Densitometry was performed using Photoshop 7 Histogram image analysis of positive regions subtracting background density in an equal area. Cell numbers were automatically counted using the Analyze Particles function of ImageJ 1.33u software, courtesy of Wayne Rasband (National Institutes of Health, Bethesda, MD). We used the t test for statistical analysis between paired sets (mutant vs. control).


   ACKNOWLEDGMENTS
 
We thank Dr. Nobuhiro Miki at the Tokyo Women’s Medical University Hospital for his gift of the antimouse GHRH antibody and Dr. Karen Sheppard for critically reading the manuscript.


   FOOTNOTES
 
This work was supported by the "Deutsche Forschungsgemeinschaft" through Sonderforschungsbereich (SFB) 405, SFB 488, Forschergruppe 302, Graduiertekolleg (GRK) 791/1.02, GRK 484 and Sachbeihilfe Schu 51/7-2, by the "Fonds der Chemischen Industrie," the European Community through Grant QLG1-CT-2001-01574, the Bundesministeriums für Bildung und Forschung; through Nationalen Genomforschungsnetzes Grant FZK 01GS011117, the Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren (HGF) through the "Strategiefonds DNA-CHIPS," the Alexander von Humboldt-Stiftung through the Max-Planck-Forschungspreis für Internationale Kooperation 1998, and the Volkswagen-Stiftung through Grant I/76 234. Part of this work was also supported by the Australian National Health and Medical Research Council.

T.M. and R.G.R. are honorary staff and S.D. is a student of the Department of Pathology, University of Melbourne.

First Published Online September 1, 2005

Abbreviations: ATF, Activating transcription factor; CRE, cAMP response element; CREB, CRE-binding protein; CREM, CRE-modulatory protein; IHC, immunohistochemical analysis; ME, median eminence; PRL, prolactin.

Received for publication May 17, 2005. Accepted for publication August 23, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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