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Endocrinology Vol. 138, No. 8 3222-3227
Copyright © 1997 by The Endocrine Society

ARTICLES

Placenta-Specific Expression of the Rat Growth Hormone-Releasing Hormone Gene Promoter in Transgenic Mice1

Núria Nogués2, José A. Del RÍo, Mercè Pérez-Riba, Eduardo Soriano, Richard A. Flavell3 and Albert Boronat

Department of Biochemistry and Molecular Biology, Faculty of Chemistry, (N.N., M.P.-R., A.B.), and the Department of Animal, Plant, and Cellular Biology, Faculty of Biology (J.A.D.R., E.S.), University of Barcelona, 08028 Barcelona, Spain; and the Howard Hughes Medical Institute and Section of Immunobiology, Yale University School of Medicine (R.A.F.), New Haven, Connecticut 06510-8023

Address all correspondence and requests for reprints to: Dr. Núria Nogués, Department of Medicine (M/C 640), University of Illinois College of Medicine, Chicago, Illinois 60612-7323. E-mail: NNogues{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of GH in the anterior pituitary. Along with many other hypothalamic hormones, GHRH is also expressed in the placenta, although its physiological role in this tissue has not yet been determined. The placental prepro-GHRH is identical to that found in the hypothalamus. However, the placental and hypothalamic GHRH messenger RNAs differ in the region corresponding to the untranslated exon 1. A combined mechanism involving the use of tissue-specific promoters and the differential splicing of exon 1 generates the mature GHRH messenger RNAs in placenta and hypothalamus. As a first step toward the localization of the regulatory elements involved in the placenta-specific expression of the GHRH gene, we have generated transgenic mice containing constructs in which potential regulatory sequences of the rat GHRH gene were fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Construct GHRH-CAT1, which contains 7.5 kilobases of flanking sequences upstream to the placental transcription start site, did not promote CAT expression in the transgenic animals. In contrast, construct GHRH-CAT2, which differs from construct GHRH-CAT1 in having additional sequences located downstream to placental exon 1, exhibited high levels of CAT expression in brain and placenta. Our results show that the sequences included in construct GHRH-CAT2 contain the cis-acting regulatory elements necessary to direct developmentally regulated and cell type-specific expression of the CAT gene in the placenta. Unexpectedly, the expression of the transgene in the brain was detected in glial cells of different areas, but not in the hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATIC growth is controlled by a complex regulatory system that includes multiple hormonal interactions. Among these control mechanisms, a predominant role is exerted by the hypothalamic-pituitary system and, in particular, by the hypothalamic neuropeptides GH-releasing hormone (GHRH), which stimulates GH synthesis and secretion in the pituitary somatotrophs (1, 2, 3, 4), and somatostatin, which inhibits GH secretion (5). GHRH also exerts a mitogenic effect on somatotrophs in vitro (6) and is thought to play an important role in the proliferation and differentiation of GH-secreting cells (7).

In addition to the hypothalamus, GHRH has been detected in extrahypothalamic sites, such as the gastrointestinal tract (8), ovary (9), testis (10), lymphocytes (11), and placenta (12, 13, 14, 15, 16, 17). In particular, it has been reported that the GHRH gene is actively transcribed in rat (14, 16) and mouse (16, 17) placenta. In both species, expression of the GHRH gene in placenta is regulated during gestation, increasing from midpregnancy to term (17, 18). However, the physiological role of placental GHRH is still unknown.

Little is known about the regulation of GHRH gene expression in placenta. The rat GHRH gene is a single copy gene (19). The GHRH precursor protein is encoded by exons 2–5, whereas exon 1 contains most of the 5'-untranslated sequences. Placental GHRH messenger RNA encodes a GHRH precursor protein identical to that found in the hypothalamus (15). Nevertheless, in both rat and mouse, the placental and hypothalamic GHRH transcripts differ in the region corresponding to the untranslated exon 1 as a result of a combined mechanism involving the use of tissue-specific promoters and the alternative splicing of exon 1 (15, 16, 17). This suggests that expression of the GHRH gene is differentially regulated in hypothalamus and placenta.

The characterization of the mechanisms controlling expression of the GHRH gene in placenta may provide insights into the biological significance of GHRH synthesis in this tissue. To date, progress in defining GHRH placenta-specific regulatory elements has been hampered by the lack of a suitable placental cell line. As a first step toward the identification of the cis-acting elements involved in the placenta-specific expression of the rat GHRH gene, we have analyzed, in transgenic mice, the expression of two constructs containing potential GHRH regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) reporter gene. The results obtained indicate that sequences located downstream of the placental exon 1 are required for the placenta-specific expression of the GHRH gene. We report that a region of the rat GHRH gene consisting of approximately 20 kilobases (kb) upstream of the GHRH translation start codon contains the cis-acting elements necessary for driving developmentally regulated and cell-specific expression in the placenta of transgenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of GHRH-CAT transgenes
pGHRH-CAT1.
An approximately 8-kb rat GHRH genomic fragment containing the placental exon 1 and its 5'-flanking region was isolated from the genomic clone {lambda}rGHRH-1 (15) by HindIII digestion and subcloned into pBluescript. This plasmid was then digested with either HindIII and XbaI or XbaI and BamHI, and the following DNA fragments were purified: a 4.4-kb HindIII-XbaI fragment, a 1.8-kb XbaI-BamHI fragment, and a 1.1-kb BamHI-BamHI fragment. Construct pGHRH-CAT1 was generated by sequential subcloning of these adjacent genomic fragments into plasmid pBLCAT3 (20). After digestion of pGHRH-CAT1 with KpnI, an 8.5-kb fragment containing placental exon 1 sequences (-195 to -40) and 7-kb of its 5'-flanking region fused to the CAT reporter gene was purified as previously described (21) and used for microinjection.

pGHRH-CAT2.
A 1-kb EcoRI genomic fragment containing GHRH exon 2 was isolated from the genomic clone {lambda}rGHRH-2 (15) (the EcoRI site at the 3'-end is located within vector sequences of {lambda}rGHRH-2) and subcloned into pBluescript. The 3'-end of the insert in the resulting plasmid was modified by excising a 0.4-kb XmaI fragment (the XmaI site at the 3'-end is located within the polylinker of pBluescript) and replacing it with a 70-bp PCR-generated fragment. The resulting insert contained 10 bp of exon 2 sequences (upstream the ATG initiator codon) and 0.6 kb of 5'-flanking region. A region further upstream was provided by a 3.2-kb XhoI-EcoRI fragment (containing hypothalamic exon 1) and a 7-kb XhoI-XhoI fragment, both isolated from {lambda}rGHRH-2 and subsequently cloned into the same plasmid. Finally, an 8.6-kb KpnI-XhoI fragment excised from {lambda}rGHRH-1 (including placental exon 1 and its 5'-flanking region) was also inserted (the 5'-end of this fragment was modified to introduce a NotI site). The CAT reporter gene and simian virus-40 polyadenylation signals were introduced downstream of the GHRH gene sequences as a 1.6-kb fragment, isolated from pBLCAT3, which was blunt end ligated to the XbaI site in the polylinker. The resulting plasmid, designated pGHRH-CAT2, was digested with NotI, and an approximately 21-kb fragment was purified and used for microinjection.

Generation of transgenic mice
Transgenic mice were generated by microinjecting fertilized (C57BL/6 x C3H) F2 mouse eggs with the constructs described above. The DNA fragments were introduced into the male pronuclei, and viable eggs were then transferred into the oviducts of pseudopregnant females (22). Transgene-positive animals were identified by slot blot analysis of tail DNA using a 32P-radiolabeled 1.6-kb CAT fragment as a probe. Four positive founder lines were established for each construct.

CAT assay
Tissue samples were dissociated mechanically by forcing through a 50-µm nylon mesh in PBS at 4 C. The resulting homogenates were centrifuged at 300 x g for 5 min at 4 C, rinsed twice with PBS, and then resuspended in 400 µl 0.25 M Tris-HCl, pH 7.5. Samples were subsequently lysed by repeated freeze-thaw cycles. Tissue lysates were heated at 55 C for 10 min and centrifuged at 10,000 x g, and the supernatants were stored frozen until assayed. Protein concentrations were determined using the Bio-Rad protein assay kit (Richmond, CA). Samples containing 100 µg protein from each tissue lysate were assayed for CAT activity using [14C]chloramphenicol (Amersham, Arlington Heights, IL; 57 mCi/mmol), as described by Gorman et al. (23).

Analysis of transgene expression in placenta during gestation
Placentas were isolated from pregnant transgenic females at different gestational stages. Minimal amounts of fetal tissues were used to extract DNA for determination of genotype by PCR, using oligonucleotide primers specific for CAT gene amplification, as described by Kesterson et al. (24). Gestational age was estimated by designating the day that the copulation plug was observed as day 0 of pregnancy. Placentas corresponding to transgenic fetuses were then assayed for CAT activity, as described above.

Immunocytochemistry
Placentas (day 17 of gestation) were fixed overnight with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4); soaked in 10%, 20%, and 30% sucrose solutions; and then snap-frozen in Tissue-Tek OCT compound (Miles Laboratories, Elkhart, IN). Sagittal and transverse cryostat sections (12-µm thick) were mounted on gelatin-coated slides and processed for the immunocytochemical detection of CAT. Briefly, sections were rehydrated in PBS-0.2% Triton X-100 and treated with 10% methanol-1.5% hydrogen peroxide to inhibit endogenous peroxidase activity. Sections were subsequently blocked with 0.2 M glycine-10% normal goat serum and then incubated with anti-CAT rabbit polyclonal antibody (5 Prime->3 Prime, Boulder, CO), diluted to 1:1000. The tissue-bound primary antibody was detected using the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, CA) (25). Peroxidase activity was developed with 0.03% diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide, and 0.2% nickel ammonium sulfate (26). Selected sections were slightly counterstained with hematoxylin, dehydrated, and mounted in DPX (Serva, Heidelberg, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of transgenic mouse lines
To define regions in the rat GHRH gene responsible for appropriate placenta-specific expression, we analyzed the expression of two constructs containing potential GHRH regulatory sequences in transgenic mice. Construct GHRH-CAT1 contains placental exon 1 sequences (nucleotides -195 to -40) and 7 kb of its 5'-flanking region fused to the CAT reporter gene (Fig. 1BGo). Construct GHRH-CAT2 consists of approximately 20 kb of genomic sequences upstream of the GHRH translation start codon (located within exon 2) also fused to the CAT reporter gene (Fig. 1BGo).



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  		Figure 1. GHRH-CAT constructs used for the generation of transgenic mice. A, Schematic representation of the rat GHRH gene. The untranslated sequences are indicated by black boxes. For simplicity, exon sizes are not drawn to scale. The genomic clones and the restriction sites used in the preparation of the constructs are also shown. B, GHRH-CAT1 and GHRH-CAT2 constructs. Construct GHRH-CAT1 contains placental exon 1 sequences (-195 to -40) and 7 kb of its 5'-flanking region fused to the CAT reporter gene. Construct GHRH-CAT2 consists of approximately 20 kb of genomic sequences upstream of the GHRH translation start codon (located within exon 2), also fused to the CAT reporter gene. The gray box located downstream of the CAT gene represents the simian virus 40 small t intron and polyadenylation signals.

 
Transgenic mice were generated by microinjecting the GHRH-CAT fusion genes into fertilized mouse eggs. Positive transgenic founders were identified by slot blot analysis. For the GHRH-CAT1 transgene, 11 of 40 progeny screened were positive, whereas for GHRH-CAT2, of 53 progeny screened, 17 were positive. Four independent transgenic lines, with 5–15 integrated copies, were established for each construct.

Tissue distribution of CAT activity in transgenic mice
Transgenic mice carrying construct GHRH-CAT1 or GHRH-CAT2 were killed, and CAT assays were conducted on tissue extracts from brain, liver, pancreas, spleen, thymus, testis, and placenta (day 17 of gestation). Nontransgenic littermates were also examined as negative control animals. No CAT activity was detected in placenta or any of the other tissues tested from mice of the four independent lines carrying the GHRH-CAT1 transgene (data not shown). In contrast, mice carrying the GHRH-CAT2 transgene exhibited CAT activity in brain and placenta (Fig. 2Go). This pattern of expression was consistently observed in the four independent lines, although the relative level of expression was considerably different from one line to another. Such quantitative differences in expression might be attributed to chromosome position effects. These results indicate that placenta-specific expression of the GHRH gene requires some element(s) located downstream of placental exon 1, present in construct GHRH-CAT2, but not in construct GHRH-CAT1.



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  		Figure 2. Pattern of GHRH-CAT2 expression in transgenic mice. An autoradiogram of the CAT assay corresponding to the analysis of CAT expression in various tissues from transgenic line 56 is shown. The CAT assay was carried out with 100 µg protein from each tissue.

 
GHRH-CAT2 transgene expression in placenta
Because of the higher levels of CAT activity observed in placentas from transgenic line 56 (10 copies integrated), we chose this line to further characterize transgene expression in this tissue. Total RNA was isolated from transgenic placental tissue and subjected to reverse transcription-PCR amplification. After complementary DNA synthesis, amplification was performed using oligonucleotide primers corresponding to rat placental exon 1 sequences downstream of the major transcription start sites (5'-CGCCTGGATCCCACAACTGC-3') and to CAT gene sequences (5'-GCATGATGAACCTGAATCGCC-3'). In agreement with appropriate placenta-specific transcription initiation and RNA processing of the transgene, a major product of 668 bp was amplified. Further confirmation of the appropriate processing was provided by sequence analysis of the 668-bp PCR product (data not shown).

To determine whether GHRH-CAT2 transgene expression is also regulated during gestation, placentas from the 13th, 15th, 17th, and 19th days of pregnancy were obtained. CAT activity levels increased progressively from midpregnancy and reached a peak on gestation day 17 (Fig. 3AGo), thus coinciding with the pattern of expression of the endogenous GHRH gene (17, 18). These results indicate that construct GHRH-CAT2 contains the regulatory elements responsible for the temporal regulation of GHRH gene expression. On the other hand, as shown in Fig. 3BGo, placentas obtained from the same transgenic female on day 17 of gestation, but whose corresponding fetuses were not transgene positive, displayed no significant CAT activity, indicating that the placental cells expressing the transgene are of fetal origin.



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  		Figure 3. GHRH-CAT2 expression in the placenta. A, Analysis of CAT expression in the transgenic placenta during gestation. One hundred micrograms of protein were assayed for each of the indicated gestation days. B, Analysis of CAT expression in placentas obtained from the same transgenic female on day 17 of gestation, whose corresponding fetuses were transgenic (1) and nontransgenic (2).

 
Trophoblast-specific expression of GHRH-CAT2 transgene
The distribution of CAT expression in the placenta of transgenic mice was analyzed by immunocytochemistry, using a rabbit polyclonal antibody anti-CAT. Nontransgenic placentas were used as a negative control. As shown in Fig. 4Go, CAT staining was detected predominantly in the basal zone of the placenta (B and D), although weak expression was also found in cells scattered throughout the labyrinth (E). Interestingly, transgene expression was detected in spongiotrophoblast cells, but not in giant cells, syncytiotrophoblasts, or decidual cells. Placental histological terminology was developed by Davies and Glasser (27).



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  		Figure 4. Immunolocalization of GHRH-CAT2-expressing cells in the mouse placenta. A–C, Low magnification photomicrographs illustrating the distribution of CAT-positive cells in transverse placental sections on day 17 of gestation. Numerous immunoreactive cells can be observed in the basal zone (BZ) of the transgenic placenta (B), whereas equivalent sections from nontransgenic control mice are devoid of immunostaining (A). C, Hematoxylin-stained section to allow area identification. D and E, High magnification photomicrographs showing CAT-positive cells within the BZ (D) and labyrinth (E). The section shown in E was slightly counterstained with hematoxylin. Scale bars: A–C = 200 µm; D = 75 µm; E = 50 µm.

 
This particular pattern of expression correlates very well with that reported for GHRH expression in the rat placenta (14). The results obtained thus indicate that the rat GHRH regulatory sequences present in the GHRH-CAT2 transgene are capable of driving cell type-specific expression of the CAT gene in mouse placental cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Along with many other hypothalamic neuropeptides, GHRH is also expressed in the placenta
Although the physiological role of placental GHRH is still unknown, it has been suggested that it can play a role in the regulation of fetal growth hormone secretion during the embryonic period (28, 29) as well as in the regulation of placental functions in a paracrine manner (30). Characterization of the mechanisms controlling the expression of the GHRH gene in the placenta may provide new insights into the biological significance of GHRH synthesis in this tissue.

With the aim of defining the regulatory elements involved in the placenta-specific expression of the rat GHRH gene, we have analyzed the expression of two constructs containing putative GHRH regulatory sequences in transgenic mice. In previous studies, the rat choriocarcinoma cell line Rcho (31) has been used as a model system to analyze the GHRH placental promoter. We performed transient transfection experiments of constructs containing different fragments of GHRH placental exon 1 5'-flanking sequences (extending from 0.3–7 kb), but none of those constructs showed expression in the Rcho cells. Thus, the results obtained suggested that control elements required for the GHRH placenta-specific expression could be located either further upstream or downstream of the placental exon 1. Nevertheless, it cannot be ruled out that under the conditions required to propagate these cells, they may not contain (or may contain in an inactive form) the transcription factors needed for GHRH gene expression. This might be the consequence of their differentiation state and/or the specific requirement of external stimuli (hormone or growth factor) essential for GHRH gene expression.

As an alternative approach, we then decided to use transgenic mice as a model system. In fact, previous studies on the trophoblast-specific expression of another placental hormone, mouse placental lactogen II, had previously been successful when using a transgenic model instead of the Rcho cells (32).

For our transgenic approach, we used two different constructs. Construct GHRH-CAT1 corresponds to the largest construct we had previously analyzed by transient transfection in the Rcho cells. Construct GHRH-CAT2 included additional sequences located downstream of placental exon 1. We considered the use of this second construct for two reasons. First, because of the genomic region included, it could allow us to analyze at the same time placenta- and hypothalamus-specific expression of the reporter gene. Second, this construct could be informative if sequences located downstream of the placental exon 1 were necessary for GHRH placenta-specific expression.

The GHRH promoter sequences present in construct GHRH-CAT1 proved to be unable to direct placental expression of the reporter gene. In contrast, mice carrying construct GHRH-CAT2 exhibited CAT activity in brain and placenta, in agreement with tissue-specific expression of the transgene. These results indicate that some element(s) important for GHRH placenta-specific expression is located downstream of the placental exon 1, within the sequences included in construct GHRH-CAT2. The involvement of intron sequences in transcriptional regulation has been previously reported in many genes. Further analysis will be necessary to localize and identify the precise sequences involved in placenta-specific expression of the GHRH gene.

Analysis of GHRH-CAT2 transgene expression in the placenta has revealed that this construct contains regulatory sequences that confer developmental regulation of the CAT gene. The pattern of GHRH-CAT2 expression throughout gestation correlates with the pattern of expression of the endogenous GHRH gene. In addition, the cellular pattern of transgene expression detected by immunocytochemistry in placental sections, also correlates with cell type-specific expression of GHRH-CAT2. The cellular pattern observed in the transgenic placenta coincides with that reported for the rat GHRH placental expression (14), which significantly differs from the mouse GHRH placental expression (17). In this respect, it is interesting to note that the cellular pattern of expression of the GHRH-CAT2 transgene is determined by the rat GHRH regulatory sequences rather than by the mouse placental cells carrying and expressing the transgene.

It is especially interesting to remark that placentas obtained from the same pregnant female heterozygotic for the transgene, only exhibited CAT expression when the corresponding fetuses were also transgenic. Otherwise, despite the fact that the maternal component of the placenta was transgenic, no expression of the transgene was detected. Thus, the placental cells expressing the GHRH gene and, in turn, the GHRH-CAT2 transgene are of fetal origin.

At present, the regulatory elements that confer hypothalamus-specific expression of the GHRH gene are not known. Identification of these elements has also been hampered by the lack of a suitable hypothalamic cell line and by the inappropriate expression of potential hypothalamic GHRH regulatory sequences in transgenic mice (33, 34). The tissue distribution of CAT expression in GHRH-CAT2 transgenic mice suggested that this construct might also contain the cis-acting elements necessary to direct expression in the hypothalamus. However, immunocytochemical analysis of CAT expression within the brain of the transgenic mice exhibited an unexpected pattern. No staining was detected in the neurons of the arcuate nuclei, where GHRH-expressing cells are predominantly localized (17, 35). In contrast, transgene expression was observed in glial cells of different areas of the nervous system, including olfactory bulb, hypothalamus, hippocampus, septum, and spinal cord (data not shown). The glial nature of the cells expressing the transgene was confirmed by double immunofluorescence with a monoclonal antibody anti-glial fibrilary acidic protein and the polyclonal antibody anti-CAT. In these studies we observed 85% colocalization (data not shown). This pattern of expression was detected in two different transgenic lines, suggesting that it is not related to chromosomal insertion. This abnormal expression of GHRH-CAT2 transgene in the brain indicates that this construct does not contain the regulatory sequences necessary for directing appropriate hypothalamic expression. One possibility is that some hypothalamus-specific element could be located at the 3'-end of the GHRH gene, which has not been tested. It is also possible that GHRH-CAT2 is indeed expressed in the arcuate nuclei, but that the level of expression is not sufficient to be detected by immunocytochemistry. As the GHRH gene is not normally expressed in glial cells, the unexpected expression of this transgene in glia might be the consequence of the malfunction of species-specific regulatory elements of the rat GHRH promoter in the mouse.

Recently, it has been described that GHRH transcription in rat testis initiates approximately 700 bp 5' to transcription initiation in placenta (36). As the newly characterized GHRH testicular exon 1 is included in both GHRH-CAT1 and GHRH-CAT2 transgenes, it was interesting to examine CAT expression in the testis of transgenic mice. Nevertheless, transgenic males carrying either GHRH-CAT1 or GHRH-CAT2 construct did not exhibit testicular expression of the CAT gene (data not shown). As in the case of the hypothalamic expression, the lack of some essential tissue-specific element(s) could explain the results obtained. Another explanation could be that the sensitivity of the CAT assay was not enough to detect transgene expression in the testis.

In conclusion, the results reported here show that the construct GHRH-CAT2 contains the GHRH regulatory elements necessary to direct developmentally regulated and cell type-specific expression of the CAT gene in the placenta. GHRH-CAT2 transgenic mice should, therefore, be an interesting model to study placenta-specific regulation of the GHRH gene in vivo.


   Acknowledgments
 
We thank Cindy Hughes and Debby Buktus for generating the transgenic mice, and David Peck and Frank Wilson for their assistance with the animal shipment. We also thank Lawrence Frohman and Rhonda Kineman for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Grant 92/0325 from the Fondo de Investigaciones Sanitarias de la Seguridad Social and Grant GRQ94–1034 from the Comissió Interdepartamental de Recerca i Innovació Tecnològica de la Generalitat de Catalunya. Back

2 Recipient of a predoctoral fellowship from the Direcció General d’Universitats de la Generalitat de Catalunya. Back

3 Investigator with the Howard Hughes Medical Institute. Back

Received February 26, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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