No abstract

Recent studies have demonstrated a clear role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropin biosynthesis and secretion, both alone and in conjunction with GnRH. First defined as a hypothalamic releasing factor, PACAP subsequently has been identified in the gonadotrope subpopulation of the anterior pituitary gland, suggesting that PACAP may act as an autocrine-paracrine factor in this tissue. In initial studies, we determined that GnRH markedly stimulated endogenous PACAP mRNA levels and promoter-reporter activity in the mature gonadotrope cell line, LbetaT2. GnRH-stimulated rat PACAP promoter activity was blunted with deletion from position -915 to -402 and eliminated with further truncation to position -77 relative to the transcriptional start site. Site-directed mutagenesis demonstrated a functional requirement for a cAMP response element (CRE)-like site at position -205 and an activating protein-1 (AP-1)-like site at position -275, both of which bound CRE binding protein and AP-1 family members on EMSA. Treatment with pharmacological activators or inhibitors of second messenger signaling pathways implicated the protein kinase A, protein kinase C, and MAPK pathways in the GnRH response. In support of these in vitro data, we demonstrate that JunB binds to the rat PACAP gene promoter by chromatin immunoprecipitation assay and that small interfering RNA knockdown of JunB, cFos, and CRE binding protein factors blunts PACAP expression. In summary, these results further elucidate the complex functional interactions between PACAP and GnRH in the anterior pituitary. Specifically, these studies demonstrate that GnRH-stimulated PACAP gene expression is mediated via multiple signaling pathways acting on CRE/AP-1 sites in the proximal gene promoter. Because both PACAP and GnRH regulate gonadotropin biosynthesis and secretion, these results provide important insight into the critical fine tuning of gonadotrope function and, thereby, the maintenance of normal reproductive function.

As endocrinologically active cells, adipocytes are capable of secreting various adipocytokines such as leptin, resistin, and adiponectin to impact metabolic function. Although adipocytes remain to be the primary site of synthesis and secretion, there is now growing evidence that supports the presence of adiponectin and its receptors within the hypothalamic-pituitary-gonadal axis, providing a possible link between obesity and abnormal reproductive physiology. It has been demonstrated that adiponectin may reduce gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus as well as modulate gonadal steroid hormone production. Furthermore, prior data indicate that adiponectin may play a role in decreasing luteinizing hormone secretion from pituitary gonadotropes. We aimed to identify the hormonal regulators of adiponectin and its receptors, AdipoR1 and AdipoR2, in pituitary gonadotropes using immortalized gonadotropic LβT2 cells and primary rat pituitary cells. Our study shows significant alterations in adiponectin expression across the estrous cycle. In addition, we present a novel finding that GnRH suppresses pituitary adiponectin expression via the calcium and protein kinase A intracellular pathways in both cultured rat primary pituitary cells and the LβT2 gonadotrope cell line. The GnRH did not alter expression of the adiponectin receptors, AdipoR1 and AdipoR2, in cultured gonadotropes. Expression of the adiponectin receptors, AdipoR1 and AdipoR2, was not altered by GnRH in cell culture but in vivo or in vitro. Our data suggest that gonadotrope function may be modulated by GnRH-mediated changes in adiponectin expression.

Here we describe our analyses of Rhino, a novel member of the Heterochromatin Protein 1(HP1) subfamily of chromo box proteins. rhino (rhi) is expressed only in females and chiefly in the germline, thus providing a new tool to dissect the role of chromo-domain proteins in development. Mutations in rhi disrupt eggshell and embryonic patterning and arrest nurse cell nuclei during a stage-specific reorganization of their polyploid chromosomes, a mitotic-like state called the “five-blob” stage. These visible alterations in chromosome structure do not affect polarity by altering transcription of key patterning genes. Expression levels of gurken (grk), oskar (osk), bicoid (bcd), and decapentaplegic (dpp) transcripts are normal, with a slight delay in the appearance of bcd and dpp mRNAs. Mislocalization of grk and osk transcripts, however, suggests a defect in the microtubule reorganization that occurs during the middle stages of oogenesis and determines axial polarity. This defect likely results from aberrant Grk/Egfr signaling at earlier stages, since rhi mutations delay synthesis of Grk protein in germaria and early egg chambers. In addition, Grk protein accumulates in large, actin-caged vesicles near the endoplasmic reticulum of stages 6–10 egg chambers. We propose two hypotheses to explain these results. First, Rhi may play dual roles in oogenesis, independently regulating chromosome compaction in nurse cells at the end of the unique endoreplication cycle 5 and repressing transcription of genes that inhibit Grk synthesis. Thus, loss-of-function mutations arrest nurse cell chromosome reorganization at the five-blob stage and delay production or processing of Grk protein, leading to axial patterning defects. Second, Rhi may regulate chromosome compaction in both nurse cells and oocyte. Loss-of-function mutations block nurse cell nuclear transitions at the five-blob stage and activate checkpoint controls in the oocyte that arrest Grk synthesis and/or inhibit cytoskeletal functions. These functions may involve direct binding of Rhi to chromosomes or may involve indirect effects on pathways controlling these processes.

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with gonadotropin-releasing hormone (GnRH). Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine-paracrine factor in this tissue. GnRH has been shown to increase pituitary Adcyap1 gene expression though the interaction of CREB and jun/fos with CRE/AP-1 cis-elements in the proximal promoter. For the current studies, we were interested in identifying additional transcription factors and cognate cis-elements which regulate Adcyap1 gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and EMSA analysis, we demonstrate that GATA2 and GATA4 stimulate Adcyap1 promoter activity via a GATA cis-element located at position −191 in the rat Adcyap1 gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GnRH-mediated stimulation of Adcyap1 gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GnRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GnRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.