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Signalisation Cellulaire, Régulation de Gènes et Physiologie de l’Axe Gonadotrope (A.G., C.B., C.G., H.P., S.M., A.T.-V., R.C., J.-N.L.), Centre National de la Recherche Scientifique Unité Mixte de Recherche 7079, Physiologie et Physiopathologie, Université Pierre et Marie Curie, 75252 Paris cedex 05, France; and Génétique, Développement et Pathologie Moléculaire (V.N.-M., D.D.), Institut National de la Santé et de la Recherche Médicale Unité 567, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut Cochin, Université René Descartes, 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. Jean-Noël Laverrière, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7079, Physiologie et Physiopathologie, Université Pierre et Marie Curie, 4 place Jussieu, Case courrier 256, 75252 Paris cedex 05, France. E-mail: jean-noel.laverriere{at}snv.jussieu.fr.
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-subunit at embryonic day 13.5 and with that of steroidogenic factor 1 at later stages of pituitary development. Transgene expression was also found in specific brain areas, such as the lateral septum and the hippocampus. A single promoter is thus capable of directing transcription in highly diverse tissues, raising the question of the different combinations of transcription factors that lead to such a multiple, but nevertheless cell-specific, expressions of the GnRH-R gene. |
Introduction |
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- and distinct ß-subunits, enter into the systemic circulation, and influence gonadal functions including gametogenesis and steroidogenesis and induce ovulation.
Modulation of the number of GnRH-Rs at the surface of gonadotrope cells is a key determinant for the fine tuning of the reproductive function, especially at the onset of puberty and throughout the estrous cycle. This regulation takes place, at least in part, at the transcriptional level. To identify the mechanisms that underlie the tissue-specific and the regulated expression of the GnRH-R gene, the 5' regulatory sequences have been isolated and partially characterized in human, mouse, rat, ovine, and porcine species (2, 3, 4, 5, 6, 7, 8). By means of transient transfection studies conducted on the well-characterized T3–1 and LßT2 mouse gonadotrope cell lines, the steroidogenic factor-1 (SF-1) and the activating protein 1 were shown to play a crucial role in the tissue-specific activity of the mouse, rat, and human promoters (9, 10, 11, 12). In addition, other factors such as Smad, LIM homeodomain-related proteins, and GATA-related factors seem to be involved in a species-specific manner (12, 13, 14). Because all of these studies have been performed in the same mouse gonadotrope-derived cell models, T3–1 and LßT2 cell lines, species-specific differences would reside in the diversity of the cis-acting elements present in the human, mouse, and rat promoters.
A prerequisite evaluation of the physiological relevance of these in vitro data led to the elaboration of mouse transgenic models. Because the different GnRH-R gene promoters preserved their tissue and species specificity in the mouse transcriptional environment in vitro, one could suppose that they could also show the same properties in vivo in transgenic mice. Thus, transgenic mouse models have been developed using not only the mouse but also the ovine promoter fused upstream of the luciferase reporter gene. The 1900-bp mouse promoter directed the expression of the luciferase reporter gene predominantly in the pituitary gland but also in brain and testes (15) in agreement with previous studies that have demonstrated GnRH-R expression in extrapituitary sites. Likewise, the activity of the 2700-bp ovine promoter was primarily confined to the pituitary gland, brain, and gonads (16). In both cases, pituitary luciferase activity was strongly reduced by passive immunization against GnRH. However, in these early works, neither the brain areas nor the pituitary cell types that expressed the transgene were identified.
To evaluate, at the cellular level, the ability of the promoter of the rat GnRH-R gene to direct expression in both pituitary and nonpituitary tissues, the 3.3-kb rat promoter was placed upstream of the human placental alkaline phosphatase (hPLAP) coding sequence. This reporter gene was chosen because it displays several interesting features. Under the control of an ubiquitous promoter, hPLAP produces a widespread and uniform pattern of marker gene expression in adult mice as well as during embryonic development, indicating that its expression is not influenced by either the type or developmental stage of the tissue. More importantly, it can be localized at the cellular level and provides simplicity and flexibility of visualization techniques as well as compatibility with most other tissue procedures (17).
Herein we show that the rat promoter directs the expression of the hPLAP transgene in pituitary cells that produce LHß and FSHß in adult mice. During pituitary development, hPLAP displays temporal and spatial expression patterns, which are consistent with those characterizing the emergence of the gonadotrope lineage. Finally, the hPLAP transgene is expressed in distinct and specific areas of the central nervous system, in agreement with the identified extrapituitary sites of GnRH-R expression.
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Materials and Methods |
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Gene construct
The construct used for transgenesis was based on the pM-AP vector (Ngô-Muller, V., and D. Daegelen, unpublished data), itself derived from the pM310CAT vector (18) in which the chloramphenicol acetyl transferase reporter gene has been replaced by the hPLAP reporter gene. Briefly, the muscle-specific promoter of the aldolase A gene (pM) was excised from the pM-AP vector and replaced by a synthetic multiple cloning site containing in the 5' to 3' direction the restriction sites for ScaI, EcoRI, HindIII, BamHI, SalI, and XhoI. The 3.3-kb HindIII/SalI DNA fragment of the rat GnRH-R gene promoter was then inserted into this new vector upstream of hPLAP coding sequence. Restriction mapping was used to confirm the identity of the plasmid. The resulting vector thus contained the rat promoter directing the expression of the hPLAP reporter gene followed by a polyadenylation signal and the SV40 intron to facilitate expression of the transgene.
Production of the transgenic mice
The transgene construct was excised from bacterial vector sequences by digestion with ScaI and HindIII and separated by agarose gel electrophoresis. The fragment was isolated and purified on Elutip column according to the manufacturer’s recommendations (Schleicher and Schuell France SARL, Mantes-La-Ville, France). Transgenic mice were produced by classic microinjection techniques. Zygotes were obtained from superovulated B6D2 F1 female mice mated with B6D2 F1 males the night before. A few hundred copies of the foreign DNA were microinjected into the male pronucleus, and eggs were reimplanted at the one-cell stage into the infundibulum of pseudopregnant foster mothers.
Southern blots
Transgenic mice were detected by Southern blot analysis. Ten micrograms of mouse tail DNA was digested with HindIII or EcoRV restriction enzymes, separated on 0.7% agarose gels and transferred onto nylon membrane (Amersham Biosciences, Orsay, France). The 3.3-kb rat GnRH-R promoter DNA fragment was labeled with digoxigenin and used as a hybridization probe according to the manufacturer’s protocol (Roche Diagnostics France S.A., Meylan, France).
Immunohistochemistry and cytochemistry
Animals were anesthetized with 3.5% chloral hydrate in PBS and perfused with 50 ml of freshly prepared 4% (wt/vol) paraformaldehyde (PFA) in PBS. Brains were further fixed overnight in 4% PFA; washed twice with PBS (pH 7.4); incubated sequentially in PBS containing 12, 15, and 18% sucrose at 4 C for 4 h each; and then finally frozen at -60 C in an isopentane bath in liquid nitrogen. Pituitary glands were fixed for 1 h at 4 C with 2% PFA in PBS; rinsed with 12, 15, and 18% sucrose in PBS for 30 min each; embedded in TissueTek OCT compound (Sakura/CML, Nemours, France); and frozen at -80 C. Fifteen-micrometer sections of embedded brain or 5 µm for pituitary gland were cut on a Reichert-Jung 2700-Frigocut cryostat. Sections were thaw mounted onto Super Frost Plus slides (Menzel-Glaser/CML, Nemours, France). Immunostaining was performed with rabbit polyclonal antibody directed against porcine LHß (no. 19526 from Dr. Y. Tillet, Nouzilly, France) (19) at a 1/200 dilution, rabbit polyclonal antirat FSHß (National Institute of Diabetes and Digestive and Kidney Diseases) at a 1/100 dilution, rabbit polyclonal antisynthetic human GH (NIDDK, no. IC-4, AFP-1613102481) at a 1/100 dilution, rabbit polyclonal antirat TSHß (NIDDK, no. IC-1, AFP-1274789) at a 1/200 dilution, polyclonal antiserum raised against bovine SF-1 (from Dr. K.-I. Morohashi) at a 1/500 dilution (20), antiovine glycoprotein hormone -subunit at a 1/200 dilution from our laboratory, and goat polyclonal antimouse GATA4 (sc-1237, Santa Cruz Biotechnology Inc., Heidelberg, Germany) at 1/200 dilution. In some cases, the sections were permeabilized with 0.05% Triton x 100 in PBS containing 0.5% BSA for 1 h before immunostaining. Incubation with the primary antibody was performed for 2 h at 37 C or overnight at 4 C. Fluorescein isothiocyanate-conjugated secondary antibodies directed against rabbit immunoglobulins were subsequently used to reveal primary antibodies. The slides were photographed, heated to 65 C to inhibit endogenous alkaline phosphatase activity, and thereafter processed for revelation of transgene activity. For this, the sections were incubated with a mixture of chromogenic enzyme substrate containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in a buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 5 mM MgCl2 for 5–20 min. The sections were then washed with PBS to block the enzymatic reaction and photographed again.
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Results |
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10 copies/genome) within the Y chromosome because it was exclusively transmitted to the male progeny. Concerning the other two lines, the transgene was integrated in autosomes as a single copy gene for line no. 1 and in multiple copies (10/genome) for line no. 17.
Transgene expression in the adult pituitary gland
The 3.3-kb rat promoter contains well-characterized cis-acting elements, such as the SF-1 response element, the activating protein 1 binding site, and the GnRH-R-specific enhancer required for full gonadotrope-specific expression in in vitro transient transfection assays (12). To evaluate the ability of this promoter to control the expression of the hPLAP transgene in vivo, transgenic adult males from each line were killed and the pituitary gland, containing the gonadotrope cells, was processed for hPLAP histochemistry. Similar results were obtained regardless of the transgenic line analyzed. A significant number of cells expressed hPLAP in the anterior pituitary lobe, whereas the posterior lobe contained no positively stained cells (not shown). Neither qualitative nor quantitative differences could be noted between the different lines. As expected, the pituitary gland from wild-type animal was negative.
The anterior pituitary gland contains five distinct hormone- secreting cell types: the lactotropes that produce prolactin, the somatotropes that secrete growth hormone, the thyrotropes that produce thyroid-stimulating hormone, the corticotropes that produce adrenocorticotropic hormone, and the gonadotropes that produce LH and FSH. The GnRH-R is known to be expressed exclusively in the gonadotrope cells in the anterior pituitary gland. To determine whether the expression of the transgene was restricted to the gonadotrope cells, tissue sections of pituitary glands originating from 9-d-old males from transgenic line no. 1 were processed for immunohistochemistry with anti-LHß and anti-FSHß antibodies before evaluating the hPLAP activity by histochemistry. As shown in Fig. 1A
, most of the cells immunoreactive for LHß were also found to express the hPLAP transgene. A limited number of cells were found positive for both FSHß and hPLAP (Fig. 1B). In contrast, somatotrope as well as thyrotrope cells revealed with anti-GH and anti-TSH antibodies, respectively, were demonstrated negative for hPLAP expression (Fig. 1C and 1D). Taken together, these data suggested that the 3.3-kb promoter was sufficient to confer gonadotrope-specific expression on the reporter gene in vivo in adult mice.
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, hPLAP was expressed in the developing pituitary at E13.5, the earliest time tested, and this expression was localized in the rostral region of the pituitary, the pars tuberalis. The pars distalis as well as the pars intermedia were negative. Using serial sections, immunohistochemical analyses with an antiserum specific for the glycoprotein hormone -subunit (-GSU) common to LH, FSH, and TSH were done. In agreement with data from Stoeckel et al. (24) the -GSU was also present in the pars tuberalis (Fig. 1F). Subsequent revelation of hPLAP activity on the same pituitary section demonstrated that transgene expression coincided with -GSU immunoreactivity, although less hPLAP-positive cells were detected (Fig. 1F). A similar immunohistochemical analysis was performed with the antibody directed against SF-1, the gonadotrope-specific transcription factor in the pituitary (25). In this case, at E13.5, the pituitary was negative, whereas numerous nuclei were clearly positive in the neighboring diencephalon (not shown). At E15.5, the hPLAP positive cells were localized in the ventral region of the pituitary with a minority of cells present in the pars distalis. The pars intermedia was negative as well as the posterior lobe. At this stage, as expected and in contrast to E13.5, SF-1 immunolabeling was intense in nuclei located in the ventral region of the pituitary and coincided with hPLAP staining (Fig. 1G). However, several cells positive for SF-1 were negative for hPLAP. This suggested that SF-1 was insufficient for GnRH-R promoter activity in vivo in mouse pituitary at this developmental stage. Alternatively, this may also reveal incomplete penetration of the transgene. Similar results were observed at E18 (Fig. 1, E and G). At P0, positive cells were scattered throughout the anterior lobe as in the adult (Fig. 1E). These results are in agreement with data from the literature showing that, in the mouse, the future gonadotrope cells are essentially concentrated in the ventral region of the anterior lobe during the early phases of pituitary ontogenesis whereas in the adult pituitary fully differentiated gonadotrope cells are rather dispersed throughout the anterior lobe (26). The 3.3-kb rat promoter of the GnRH-R gene thus directs transgene expression with spatiotemporal patterns analogous with those characterizing the emergence of the gonadotrope lineage. The colocalization of the transgene expression with either the -GSU at E13.5 or SF-1 at E15.5 and E18 supports this hypothesis.
Transgene expression in the brain
Because GnRH-R gene expression has been demonstrated in numerous tissues other than the pituitary gland, especially the central nervous system, this tissue was evaluated as alternative sites of transgene activity (see review in Ref.27, 28). Frontal sections of the entire brain of adult males were thus subjected to histochemical analysis (Fig. 2). The lateral septum was intensely stained, indicative of a robust site of transgene expression (Fig. 2A). Fibers were strongly positive. Simultaneous staining with 4',6-diamidino-2-phenylindole (DAPI) indicated that the unlabeled areas corresponded to cell nuclei (Fig. 3A). In more posterior regions, the transgene was also strongly expressed in the hippocampus, notably in fibers of the stratum oriens and stratum radiatum, which corresponded to the dendritic compartment of the adjacent pyramidal cell layer (Figs. 2B and 3B). Interestingly, the fiber layer located between the stratum radiatum and the pyramidal cell layer, which probably corresponded to the stratum lucidum, was unlabeled as well as the pyramidal cells. Fibers located within the fimbria hippocampi but not those emerging from the dentate gyrus stained positive for hPLAP. Staining with cresyl violet that labeled the cell nuclei contrasted with the absence of hPLAP staining in the cell bodies of the pyramidal cell layer. Areas positive with cresyl violet were negative for hPLAP staining and vice versa (not shown). Marked hPLAP staining was also detected in the amygdala and the rhinal sulcus.
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). The three-dimensional reconstruction showed a continuous labeling from the lateral septum to the posterior hippocampus, strongly suggesting the existence of functional connections in the expression of the transgene. In contrast, the rhinal sulcus and the amygdala appeared as independent labeled structures with regards to transgene expression, at least within the limits of the detection method.
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). Altogether, these localizations were consistent with, and complement, those previously reported using either in situ hybridization with a radiolabeled GnRH-R cDNA probe or after binding of the radiolabeled ligand GnRH (for review see Ref.28). Data from our group and other studies have been summarized in Table 1 (29, 30, 31). The spatial pattern of the transgene expression in the brain appeared analogous to that obtained with the binding of radiolabeled GnRH. In particular, in the hippocampus, hPLAP staining filled the fibers similarly to the ligand probe, whereas the cDNA probe labeled the pyramidal cell layer and the cell nuclei of the dentate gyrus. The pattern of transgene expression thus mimicked closely the localization of receptor protein rather than the site of mRNA expression, most likely because the transgene encoded for a glycosylphosphatidylinositol-anchored membrane bound protein. Interestingly, the lateral septum was the site for both transgene activity and GnRH binding, but not mRNA expression, strongly suggested that GnRH-R synthesis likely occurred in different brain area. As already proposed by Jennes and Woolums (29) for the GnRH-R protein in the lateral septum, the hPLAP might be located on axon terminals belonging to neurons of areas CA1 and CA3 of the Ammon’s horn, which are known to project into the lateral septum (32). This hypothesis is also in agreement with the three-dimensional reconstruction (Fig. 4).
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, A and B) but not with TSH immunostaining (Fig. 6C). The results were different in the brain. In one mouse line analyzed, although transgene expression was present in both the lateral septum and the hippocampus, the hPLAP staining was different from that obtained with the 3.3-kb promoter (not illustrated). Indeed, additional staining was observed in the fibers of the dentate gyrus. In contrast, in the second transgenic mouse line, the septum and the hippocampus were both unlabeled. These divergent results obtained using the same construct suggested that the site of transgene insertion together with the 2.2-kb deletion in the 5' upstream domain of the rat GnRH-R were decisive parameters in the brain. Analyses of other transgenic mouse lines bearing the 1.1-kb promoter linked to the hPLAP reporter gene are in progress to delineate their respective implications.
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Discussion |
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). Transgene expression is actually restricted to cells that expressed the LHß and/or FSHß subunits. In addition, ontogenetic patterns of transgene expression similarly correlate with the spatial and temporal emergence of the gonadotrope lineage during pituitary development (Fig. 2A). Indeed, in the earliest stages, the expression is restricted to the rostral region of the developing gland in which endocrine precursor cells that express the -GSU gene are typically situated. At intermediate stages, the cells expressing the transgene are detected in the ventral region of the anterior pituitary gland, the region in which the presumptive gonadotrope precursors arise under the influence of ventral BMP2 signal that directs the expression of the zinc finger protein GATA2 (33). Lastly, in the mature gland, transgene expression loses its spatial restriction in agreement with the scattered localization of fully differentiated gonadotrope cells. Therefore, in the adult as well as during ontogenesis, transgene expression driven by the 3.3-kb rat promoter probably reveals the actual site of GnRH-R gene expression. The presence of the orphan nuclear receptor SF-1 in cells expressing the transgene during pituitary development at E15.5, E18 (Fig. 2B), and later (not shown) is consistent with this hypothesis. The present data also show that the 3.3-kb pituitary promoter is active in some brain areas. Three alternate hypotheses may account for this situation. First, we cannot exclude that the unusual and species-specific mouse transcriptional environment leads to aberrant and ectopic activation of the rat promoter. However, this is improbable because several data from both mouse and rat as well as from other species support such an extrapituitary activation of the rat promoter. In particular, transcription factors likely to stimulate rat promoter activity are present in these diverse tissues. SF-1 is one of the best candidates because it is known to be expressed in several brain regions, notably the ventromedial nucleus (VMH) of the developing and adult hypothalamus in the rat (34). In newborn SF-1 knockout mice, the VMH is absent, indicating that it is essential for development of VMH neurons (35, 36). SF-1 was also shown to be expressed in marmoset and rat hippocampus, within the pyramidal cell layer and in the dentate gyrus (37). It is possible that a single transcription factor, SF-1, could be responsible for the GnRH-R promoter activity in the hippocampus, hypothalamus, and pituitary gland. In addition to SF-1, the rat GnRH-R promoter contains functional response elements for LIM homeodomain- and GATA-related factors that are required for the full promoter activity in gonadotrope cells. Such factors might be involved in the activity of the rat promoter in extrapituitary tissues also. Among the possible candidates, the LIM homeodomain protein, Lhx5, is a determinant in the development of the hippocampus (38). Indeed, in adult Lhx5 knockout mice, the pyramidal cell layer in Ammon’s horn and the granule cell layer in the dentate gyrus are absent or poorly defined. Furthermore, behavioral phenotyping of Lhx5 null mutants demonstrates that these mice display defects in learning and memory tasks (39). The LIM homeodomain protein Lhx5 is likely to be involved in hippocampus-specific gene expression and thus may be a potential candidate in mediating the hippocampal expression of the GnRH-R gene.
Alternatively, we may consider that the ability of the promoter to direct expression in multiple sites may be the consequence of distinct and specific combinatorial codes of transcription factors in each tissue. They may be identified by means of transient transfection experiments using cultured cells derived from each tissue or through transgenic mice bearing various promoter constructs. Such a study is currently under investigation in our laboratory using hPLAP fused to deleted or artificial GnRH-R gene promoters containing a restricted number of response elements. Furthermore, these codes may vary depending on the developmental stage. In the present study, the transgene was detected at E13.5 in the developing pituitary although SF-1 was not yet detected. In addition, some SF-1 positive cells at E15.5 and E18 do not express the transgene. This contrasts with data derived from transient transfection studies, which have demonstrated a pivotal role for SF-1 in mouse, rat, and human GnRH-R promoter activity (9, 11, 12, 40). Our in vivo data indicate that signals other than SF-1 are necessary for turning on the GnRH-R gene. This is in agreement with other in vivo data from SF-1 knockout mice in which gonadotropin secretion can be induced by treating animals with GnRH, indicating that the GnRH-R is present and functional despite the absence of SF-1 in these mice (35, 41).
Although the functional role of the GnRH-R gene at the pituitary level is well understood, its function and importance in other tissues yet remains a matter of debate. In the hypothalamus, the receptor likely plays a facilitatory role in sexual behavior, notably in lordosis in the rodent female (42, 43). In the hippocampus, however, its role is yet unknown even if the number of GnRH-R has been shown to be regulated either under physiological or experimental situations (44, 45). The hippocampus is known to be involved in a variety of behavioral functions, including learning and memory tasks, some of which are dependent on sexual maturity (46, 47). Under physiological conditions such as the estrous cycle and puberty, the pulsatile secretion of GnRH is also profoundly modified resulting in, for example, an acute alteration of gonadotropin secretion by the pituitary gland. It cannot be excluded that these modifications in GnRH secretion also alter the function of the GnRH-R within the central nervous system. From the location of immunoreactive GnRH, it may be deduced that the decapeptide may attain most of the brain areas expressing the GnRH-R with the exception of the hippocampus (review in Ref.27). In the latter case, it may be speculated that GnRH is released into the cerebrospinal fluid and transported through the ventricular system (27, 48) to the strata oriens and radiatum of the hippocampus, thus modifying the activity and/or the property of the hippocampal GnRH-R expressing neurons. This could result in sex behavioral difference or sex-dependent synapse plasticity.
Results of this study also demonstrate the high level of sensitivity reached using the hPLAP as a reporter gene. Indeed, during cytochemical processing, transgene expression is detected within a few minutes, and the reaction products remain confined within the cell boundaries, allowing an unprecedented and accurate analysis of the GnRH-R gene expression. Considering that the specificity and/or the affinity of available antibodies directed against the GnRH-R remain to be validated, our transgenic model offers an alternate method to study the localization and ontogenetic appearance of the GnRH-R.
In addition to the presented new features of the rat GnRH-R promoter activity in vivo, this study is the first that provides direct evidence for gonadotrope localization of a GnRH-R transgene. To date, the 1.1-kb promoter is the shortest GnRH-R promoter sequence identified that confers gonadotrope-specific expression in the pituitary. These in vivo data correlate completely with our in vitro results obtained by transient transfection of T3–1 and LßT2 cell lines (7, 12). Finally, the data obtained in the brain represent a significant step forward from previous work, especially considering the cellular localization of the transgene expression. Indeed, the continuous labeling of the nerve fibers all along the hippocamposeptal complex illustrated by the three-dimensional reconstruction strongly suggests that transgene expression follows a particular neuroanatomical connection as previously described between the hippocampal pyramidal cells and the lateral septum (32). Such descriptive information generates new insights into the multiple, but nevertheless cell-specific, sites of expression of the GnRH-R gene and lays the basis for further physiological and molecular studies.
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Acknowledgments |
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Footnotes |
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A.G. and V.N.-M. contributed equally to this work.
Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; E, embryonic day; GnRH-R, GnRH receptor; -GSU, glycoprotein hormone -subunit; hPLAP, human placental alkaline phosphatase; PFA, paraformaldehyde; SF-1, steroidogenic factor-1; VMH, ventromedial nucleus.
Received July 15, 2003.
Accepted for publication October 21, 2003.
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References |
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-subunit in the pars tuberalis of the rat pituitary gland during ontogenesis. Neuroendocrinology 58:616–624[Medline]
-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885[Abstract]
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