Identification and analysis of two novel sites of rat GnRH receptor gene promoter activity: the pineal gland and retina.

BACKGROUND AND AIMS: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. METHODS: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. RESULTS: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. CONCLUSION: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

GATA2-induced silencing and LIM-homeodomain protein-induced activation are mediated by a bi-functional response element in the rat GnRH receptor gene.

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH ß-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

Mechanisms underlying the tissue-specific and regulated activity of the Gnrhr promoter in mammals.

The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.

Reporter transgenic mouse models highlight the dual endocrine and neural facet of GnRH receptor function.

In the pituitary of mammals, the GnRH receptor (GnRHR) plays crucial roles in the neuroendocrine control of reproductive function. This receptor is specifically expressed by the gonadotrope cells scattered among the five other endocrine cell types constituting the anterior pituitary; it is also expressed in other organs, such as the gonads and brain where its function is not well defined. To gain insight into GnRHR function, distribution, and regulation, several transgenic approaches have been developed using a range of reporter genes under the control of the mouse, rat, or ovine GnRHR gene (Gnrhr) promoters. Comprehensive reviews of the literature, together with recent results obtained in our laboratory, illustrate how these transgenic models highlight the endocrine as well as the neural facet of GnRHR function. In this review, the endocrine aspect will be discussed with regard to the pituitary and gonad function, whereas the neural aspect will be discussed with regard to hippocampal formation and the oculomotor pathway, the latter constituting an unpreviously described site of Gnrhr promoter activity. These approaches should help elucidate the properties of the mammalian GnRH system.

GnRH receptor gene expression in the developing rat hippocampus: transcriptional regulation and potential roles in neuronal plasticity.

In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH) which activates specific receptors (GnRHR) present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to function as an original unit.

What is the role of PACAP in gonadotrope function?

Strong evidence in favor of a direct action of hypothalamic PACAP at the pituitary to modulate gonadotrope function has been acquired mainly by in vitro studies using cultured pituitary cells or gonadotrope cell lines. In particular, PACAP has been shown to cooperate with GnRH, the primary regulator of gonadotropes, to regulate/modulate gonadotropin subunit gene expression, gonadotropin release as well as gonadotrope responsiveness. These effects of PACAP appear to be due essentially to its high potent stimulatory action on the cAMP/protein kinase pathway. Ensuing mechanisms include signaling cross-talk and/or enhanced gene expression within gonadotropes. PACAP may also indirectly operate on these cells through paracrine mechanisms. While PACAP has long been viewed as a hypophysiotropic factor, a locally produced PACAP has also been described. Interestingly, both appear similarly up-regulated at proestrus of the reproductive cycle in female rats. Further in vivo investigation is now necessary to ascertain the physiological relevance of the observed pituitary PACAP effects and especially to evaluate the respective contribution of hypothalamic and pituitary PACAP in the dynamic control of gonadotrope function.

The LIM-homeodomain proteins Isl-1 and Lhx3 act with steroidogenic factor 1 to enhance gonadotrope-specific activity of the gonadotropin-releasing hormone receptor gene promoter.

The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.

Gonadotropin-releasing hormone and the control of gonadotrope function.

Normal gametogenesis and steroidogenesis is highly dependent on the pulsatile release of hypothalamic GnRH that binds high-affinity receptors present at the surface of pituitary gonadotrophs thereby triggering the synthesis and release of the gonadotropins LH and FSH. The mammalian GnRH receptor displays the classical heptahelical structure of G protein-coupled receptors with, however, a unique feature, the lack of a C-terminal tail. Accordingly, it does not desensitise sensu stricto, and internalises very poorly. It is now well established that GnRH stimulation induces the activation of a complex network of transduction pathways involved in the control of gonadotropin release and subunit gene expression. Other authors and ourselves have demonstrated that the GnRH action is associated with an increased complexity regarding gene regulation/cell function. Indeed GnRH affects the GnRH receptor gene itself and a number of additional genes that include some involved in cell signalling and auto-/paracrine regulation. The fact that GnRH regulates the expression of its own receptor, together with a host of other genes typically involved in its signal transduction cascades implies alteration/auto-adaptation in gonadotropic responsiveness. Furthermore, some of these genes respond differentially depending on whether the GnRH stimulation is intermittent or permanent suggesting specific roles in the dual process of activation/desensitisation. Thus, it can be assumed that the importance of pulsatility of GnRH action is closely related to, or dependent on, the inability of the GnRH receptor to desensitise. Moreover, multiple post-receptor events are crucial for both the regulation/plasticity of gonadotropic function and the maintenance of cell integrity.

[An ambiguous role of steroidogenic factor 1 in the rat GnRH receptor gene expression. Lessons from transgenic mice]. / Le rôle ambigu du facteur de transcription SF-1 dans l'expression génique du récepteur de la GnRH. Leçons de la transgenèse.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

The promoter of the rat gonadotropin-releasing hormone receptor gene directs the expression of the human placental alkaline phosphatase reporter gene in gonadotrope cells in the anterior pituitary gland as well as in multiple extrapituitary tissues.

Previous studies dealing with the mechanisms underlying the tissue-specific and regulated expression of the GnRH receptor (GnRH-R) gene led us to define several cis-acting regulatory sequences in the rat GnRH-R gene promoter. These include functional sites for steroidogenic factor 1, activator protein 1, and motifs related to GATA and LIM homeodomain response elements as demonstrated primarily in transient transfection assays in mouse gonadotrope-derived cell lines. To understand these mechanisms in more depth, we generated transgenic mice bearing the 3.3-kb rat GnRH-R promoter linked to the human placental alkaline phosphatase reporter gene. Here we show that the rat GnRH-R promoter drives the expression of the reporter gene in pituitary cells expressing the LHbeta and/or FSHbeta subunit but not in TSHbeta- or GH-positive cells. Furthermore, the spatial and temporal pattern of the transgene expression during the development of the pituitary was compatible with that characterizing the emergence of the gonadotrope lineage. In particular, transgene expression is colocalized with the expression of the glycoprotein hormone alpha-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.

Cloning and modeling of CD8 beta in the amphibian ambystoma Mexicanum. Evolutionary conserved structures for interactions with major histocompatibility complex (MHC) class I molecules.

Mammalian and avian T-cells exhibit a large number of well characterized surface molecules associated with their maturation degree. Very little is known in comparison with T-cell differentiation in ectothermic vertebrates. This is mainly due to the lack of probes to identify T-cell subsets. We cloned and sequenced the first ectothermic CD8 beta DNA complementary to RNA from an amphibian species, the Mexican axolotl. The CD8 beta chain was 30-36% identical with its avian and mammalian homologues. The extracellular V-like domain contained the two typically conserved cysteines and was followed by a J-like sequence containing the canonical Phe-Gly-X-Gly stretch. The connecting peptide was much longer than in other species and contained potential O-glycosylation sites. The axolotl CD8 beta and major histocompatibility complex class I molecules were modeled using human HLA-A2/CD8 alphaalpha complex as template. The backbone conformation of axolotl CD8 beta matched well with the CD8 alpha-2 subunit of the human complex but significant structural differences were located in the CDR1, CDR2 and DE loops. Both axolotl and human class I showed large negative surface potential. The interacting area of the human CD8 alpha chain and of the corresponding region of axolotl CD8 beta had positive electrostatic potential compatible with complexation with the corresponding class I molecules. The presence of a CD8 beta homologue in an amphibian species implies that it was already present in the Devonian ancestor of amphibians and mammals, i.e. more than 400 million years ago.