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.

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.

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.