Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.

GnRH signaling regulates reproductive physiology in vertebrates via the hypothalamic-pituitary-gonadal axis. In addition, GnRH signaling has been postulated to act on the brain. However, elucidating its functional role in the central nervous system has been hampered because of the difficulty in identifying direct GnRH signaling targets in live brain tissue. Here we used a binary genetic strategy to visualize GnRH receptor (GnRHR) neurons in the mouse brain and started to characterize these cells. First, we expressed different fluorescent proteins in GnRHR neurons and mapped their precise distribution throughout the brain. Remarkably, neuronal GnRHR expression was only initiated after postnatal day 16, suggesting peri- and postpubertal functions of GnRH signaling in this organ. GnRHR neurons were found in different brain areas. Many GnRHR neurons were identified in areas influencing sexual behaviors. Furthermore, GnRHR neurons were detected in brain areas that process olfactory and pheromonal cues, revealing one efferent pathway by which the neuroendocrine hypothalamus may influence the sensitivity towards chemosensory cues. Using confocal Ca(2+) imaging in brain slices, we show that GnRHR neurons respond reproducibly to extracellular application of GnRH or its analog [D-TRP(6)]-LH-RH, indicating that these neurons express functional GnRHR. Interestingly, the duration and shape of the Ca(2+) responses were similar within and different between brain areas, suggesting that GnRH signaling may differentially influence brain functions to affect reproductive success. Our new mouse model sets the stage to analyze the next level of GnRH signaling in reproductive physiology and behavior.

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to GnRH receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, but GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHRexpressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show that luteinizing hormoneexpressing gonadotropes, but not follicle-stimulating hormoneexpressing gonadotropes, express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in luteinizing hormone secretion is needed to promote development of follicle-stimulating hormone-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling.gonadotropin-releasing hormone neurons | gonadotropin-releasing hormone receptor | gonadotrope development | diphtheria toxin | Cre recombinase R eproductive physiology in mammals is centrally regulated through the hypothalamic-pituitary-gonadal axis and depends on gonadotropin-releasing hormone (GnRH). GnRH signaling is well understood in adult animals, but has also been implicated in the development of the reproductive axis. The hormone is produced by a subset of neurons with a scattered distribution throughout the basal forebrain and released into the hypophyseal portal vasculature from axon terminals at the median eminence (1, 2). GnRH binds to the GnRH receptor (GnRHR), which is specifically expressed on gonadotrope cells in the anterior pituitary gland (3). GnRH signaling controls biosynthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that in turn regulate development and activity of the ovaries and testes (4).Studies in mice suggest that the hypothalamic-pituitarygonadal axis might already be functional during embryonic development. GnRH neurons have migrated from their place of origin in the nasal placodes to their final destination in the forebrain (5) and project neurosecretory axons to the median emi...

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Tobramycin is an important broad spectrum aminoglycoside antibiotic widely used against severe Gram-negative bacterial infections. It is produced by base-catalyzed hydrolysis of carbamoyltobramycin (CTB) generated by S. tenebrarius. We herein report the construction of a genetically engineered S. tenebrarius for direct fermentative production of tobramycin by disruption of aprK and tobZ. A unique putative NDP-octodiose synthase gene aprK was disrupted to optimize the production of CTB, resulting in the blocking of apramycin biosynthesis and the obvious increase in CTB production of aprK disruption mutant S. tenebrarius ST316. Additional mutation on the carbamoyltransferase gene tobZ in S. tenebrarius ST316 generated a strain ST318 that produces tobramycin as a single metabolite. ST318 could be used for industrial fermentative production of tobramycin.