Veronica J. Roberts

Pax6 is implicated in murine pituitary endocrine function

Pax6, an evolutionarily conserved transcription factor, is expressed in the murine and zebrafish embryonic pituitary, but its role in pituitary development and endocrine function has not been described. To study the role of Pax6 in vivo, we examined Pax6 mutant mouse (SeyNeu) pituitaries. Mice homozygous for the SeyNeu mutation die at birth; therefore, we examined peptide hormone expression by the differentiated pituitary cell types as well as developmental marker expression in the intermediate and anterior lobes of the embryonic pituitary. GH- and PRL-immunopositive cells appear severely decreased in an outbred ICR background at embryonic d 17.5, although mRNA expression of these peptide hormones is present, as is expression of other pituitary markers. This suggests that pituitary cell types are able to differentiate in mutant embryos. To identify the cellular or physiologic mechanism responsible for less GH-and PRL-immunoreactivity in Pax6 mutant mice, we tested serum levels of GH and PRL. Pax6 homozygous mutant mice have GH serum levels one fifth that of controls at embryonic d 17.5, and one-third that of controls at postnatal d 0. PRL serum levels, which are very low during embryonic and neonatal stages, were below assay detection limits in both the wild-type and mutant groups. Taken together, these data suggest that Pax6 is not essential for pituitary differentiation, but rather functions to establish appropriate neonatal homeostatic levels of GH and PRL, possibly through regulation of translational or secretory mechanisms.

Hybridization histochemical and immunohistochemical localization of inhibin/activin subunits and messenger ribonucleic acids in the rat brain.

Inhibin and activin are best known as gonadal glycoprotein hormones but have a broad anatomical distribution. We previously described the central distribution of inhibin/activin βA‐ and βB‐subunit proteins in some neuronal cell bodies, fibers, and nuclei of the rat brain and reported a possible role for central activin in suckling‐induced oxytocin secretion and corticotropin releasing factor release. In the present report, we mapped the detailed immunohistochemical localization of inhibin/activin α‐, βA‐, and βB‐subunits throughout the rat brain to further clarify their central distribution. In addition, the localization and distribution of their corresponding mRNAs was assessed. The results are summarized as follows: 1) Both βA‐ and βB‐subunit immunoreactivity are found in neuronal cell bodies in the nucleus of the solitary tract and the dorsal and ventral medullary reticular nuclei, and in fibers and terminals of known projection sites for these nuclei. 2) βB‐subunit immunoreactivity is localized in a group of perifornical neurons in the hypothalamus. 3) βA‐subunit immunoreactivity is present in discrete populations of neuronal cell nuclei scattered throughout the CNS. 4) mRNAs encoding each of the inhibin/activin subunits are expressed in all major brain regions as determined by S1 nuclease assay and in a variety of specific neuroanatomical sites as shown by in situ hybridization. The results suggest that central inhibin and activin proteins are produced in the brain where they may potentially serve inter‐ and intracellular functions in multiple systems.

Localization of Pbx1 transcripts in developing rat embryos.

Recently, a new family of homeodomain proteins has emerged, that includes extradenticle, ceh-20, Pbx1, Pbx2 and Pbx3. The Pbx family has been shown to modulate the biological activities of the Hox proteins. We demonstrate here by in situ hybridization that Pbx1 transcripts are present in many embryonic tissues. Highest levels of Pbx1 expression in the developing embryo, from 12 to 20 days post coitum, are found in neuronal tissues, including brain, spinal cord and ganglia. In addition, Pbx1 transcripts are also detectable in the gut, lung, olfactory epithelium and kidney. The expression pattern of Pbx1 overlaps with that of many of the Hox gene products and is consistent with them acting in parallel to regulate common target genes.

Activin A regulation of gonadotropin-releasing hormone synthesis and release in vitro.

Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.

Tissue-Specific Expression of Inhibin/Activin Subunit and Follistatin mRNAs in Mid- to Late-Gestational Age Human Fetal Testis and Epididymis

Inhibin/activin subunit (α, βA, and βB) immunoreactive protein localization patterns and cell type specific inhibit α-subunit mRNA expression have been examined in early-to midgestational age human fetal testes. The scarcity of available third trimester human fetal tissue has, however, prevented a complete examination throughout the gestational period and the cell specific expression of follistatin and βA- and βB-subunit mRNAs are currently unknown at any gestational age. In the present study, this gap is filled and report mRNA expression patterns of inhibin/activin subunits in mid- and late-gestational age (21–33 wk) human fetal testes and testicular duct system. We also report the first examination of follistatin mRNA signals in the human fetal gonad is also reported. Inhibin/activin α-subunit mRNA signal is present in both tubular and interstitial cells, and βB-subunit mRNA is expressed in seminiferous tubules, in mid- and late-gestational age human fetal testes. Inhibin/activin βA-subunit mRNA was detected in the interstitial cells of remarkably well preserved mid (21 and 22 wk) and late (29 wk) gestational age testis, and is the only activin-system factor mRNA also expressed in tissue of the duct system of the testis (smooth muscle cells of the epididymis). Follistatin mRNA signal was equal to background levels in testicular and duct tissues at all ages examined. These cell specific expression patterns suggest prominent and possibly differential roles for the inhibins and activins, unopposed by gonadal follistatin, in the human fetal male reproductive system.

Expression of activin and follistatin in the rat hypothalamus: anatomical association with gonadotropin-releasing hormone neurons and possible role of central activin in the regulation of luteinizing hormone release.

The central role of activin in the regulation of the reproductive axis remains largely unexplored. Evidence suggests that activin may play a role in controlling gonadotropin-releasing hormone (GnRH) release. We assessed potential neuroanatomical associations between activin- and GnRH-neuronal systems via examination of the distribution of activin βA-subunit and activin binding protein (follistatin) protein and mRNA signals relative to GnRH neurons in the adult rat brain. Activin βA-subunit-immunostained fibers were distributed throughout the hypothalamus and GnRH-positive perikarya, and fibers were in close association with βA-subunit-immunoreactive fibers. Follistatin mRNA-expressing cells were also identified throughout the hypothalamus with GnRH fibers often observed juxtaposed to follistatin cell bodies. Colocalization of either the βA-subunit or follistatin within GnRH neurons was not detected. The functional significance of central activin in the regulation of the reproductive axis was also demonstrated. The intracerebroventricular infusion of rh-activin A significantly increased luteinizing hormone, but not follicule-stimulating hormone, serum levels in adult male rats. Taken together, the present results support an interaction between activin and GnRH neuronal systems in the rat hypothalamus, and suggest activin may act within the brain to regulate the reproductive axis.

Inhibin: role and secretion in the rat.

Publisher Summary This chapter discusses the role and secretion of inhibin in the rat. The presently known mechanisms controlling luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion include gonadotropin-releasing hormone (GnRH), sex steroids, opiates, various neurotransmitters and peptides, inhibin, activin, and follistatin. Inhibin—a molecule whose role was originally restricted to the specific inhibition of FSH secretion—is now recognized as an essential regulator of pituitary and gonadal function through complex effects of its own and interactions with other secretagogues, for example, activin, follistatin, Gns, sex steroids, and GnRH. In the female endogenous, inhibin appears to regulate both pituitary and gonadal activity. In the adult male (rat), the role of the protein might be restricted to testicular function. Despite numerous attempts toward purification and characterization of inhinbin, its structure has remained elusive. As a result of improvements in chromatography, protein sequence, and molecular cloning, a 32 kDa protein was isolated and characterized from ovine and bovine follicular fluids with the biological activities expected of inhibin. Inhibin has an inhibitory action on fertility. The role of endogenous inhibin in modulating FSH secretion in the male rat is restricted to the early part of sexual development. Immunoneutralization of endogenous inhibin increases plasma FSH levels of young male rats, and this observation suggests a functional relationship between inhibin and FSH secretion, which takes place until the relatively high plasma FSH levels characteristic of the adult male rat are established.