O órgão vomeronasal de 10 machos e 10 fêmeas de ratos Wistar adultos, com 120 dias de idade, com pesos entre 210 e 318 gramas, foram utilizados para o presente estudo com objetivo de determinar a estrutura histológica e as dimensões deste órgão para verificar a existência de dimorfismo sexual. Após perfusão, os órgãos foram imersos em solução de glutaraldeído 2,5% em tampão fosfato para fixação, isolado juntamente com o septo nasal, imerso em solução de Perenyi para descalcificação e, em seguida cortados segundo técnicas clássicas de estudos morfométricos e estereológicos. As secções foram colocadas sobre lâminas, coradas com azul de toluidina e analisadas em microscopia de luz. Também foi feita diafanização do órgão com posterior coloração com hematoxilina ácida. Foram descritas e quantificadas a constituição e distribuição dos tecidos que compõem o órgão vomeronasal de ratos machos e fêmeas, maduros sexualmente, e estabelecidas a área total de vasos, o volume do órgão, a proporção de volume ocupada pelos vasos no volume total do órgão, área total do lúmen, do epitélio sensorial e não sensorial em ratos que já atingiram a maturidade. Apesar de não existir diferenças significativas no volume do órgão vomeronasal entre machos e fêmeas de ratos Wistar adultos, os componentes deste complexo órgão, apresentam notáveis diferenças. / The vomeronasal organ of 10 male and 10 female Wistar rats, adults, 120 days weighting between 210 and 318g, were used for this study to determine the histological structure and to verify the existence of sexual dimorphism. After perfusion, the organs were fixed by immersion in 2,5 glutaraldehyde in phosphate-buffered solution; isolated with the nasal septum, immersed in Perenyi solution for decalcification and then sectioned using classic morphometric and stereologic techniques. The sections qere placed on slides and stained with toluidina blue and analysed in light microscope. Diafanization of the organ was done and this sample was stained with acid hematoxilin. The constitution and distribution of the VNO tissue of male and female rats, sexually mature were described and quantified. We also established the vessel total area, total volume organ, the volume ratio occupied by the vessels in the total volume organ, total lumen area and total sensorial and no sensorial area. However there are no significative differences in the organ volume between male and female Wistar rats, the structures of this complex organ show remarkable differences.

<p>Pheromones are chemicals from conspecifics that affect innate behavior or hormonal changes. In mammals, the vomeronasal organ (VNO) is thought to play a prominent role in detecting pheromones; the vomeronasal sensory neurons (VSNs) express three families of seven-transmembrane G-protein coupled receptors (GPCRs): the V1Rs, V2Rs, and FPRs, in two molecularly and spatially-distinct regions. In mice, VSNs that express the V2Rs are thought to detect peptide cues, including MHC-presenting peptides, major urinary proteins (MUPs), and exocrine gland-secreting peptides (ESPs). They are thought to be involved in various pheromone-mediated behaviors and physiological changes, such as mating, aggression, and selective pregnancy block. In order to understand how pheromones are detected by the vomeronasal receptors, it is essential to know which receptors are activated by a given chemical. However, identifying cognate ligands for the V2Rs has been challenging, partly because they are poorly localized to the surface of heterologous cells. Here, we show that the calreticulin chaperone family members play a crucial role in trafficking V2Rs. A calreticulin homologue, calreticulin4 is specifically expressed in the VNO, while calreticulin expression level is low. Depleting calreticulin expression in HEK293T cells allows V2Rs to be trafficked to the cell surface, whereas expression of calreticulin4 does not block the trafficking of the V2Rs. Using this knowledge, we have established a heterologous cell system to functionally identify the V2Rs and demonstrate that the ESP family members can differentially activate the V2Rs. We also show the large extracellular domain of the V2Rs plays a crucial role in ligand selectivity. Our results provide a platform to characterize ligand selectivity of the V2Rs and suggest that a unique mechanism involving calreticulins regulates the functional expression of the V2Rs.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-111).

O órgão vomeronasal de 10 machos e 10 fêmeas de ratos Wistar adultos, com 120 dias de idade, com pesos entre 210 e 318 gramas, foram utilizados para o presente estudo com objetivo de determinar a estrutura histológica e as dimensões deste órgão para verificar a existência de dimorfismo sexual. Após perfusão, os órgãos foram imersos em solução de glutaraldeído 2,5% em tampão fosfato para fixação, isolado juntamente com o septo nasal, imerso em solução de Perenyi para descalcificação e, em seguida cortados segundo técnicas clássicas de estudos morfométricos e estereológicos. As secções foram colocadas sobre lâminas, coradas com azul de toluidina e analisadas em microscopia de luz. Também foi feita diafanização do órgão com posterior coloração com hematoxilina ácida. Foram descritas e quantificadas a constituição e distribuição dos tecidos que compõem o órgão vomeronasal de ratos machos e fêmeas, maduros sexualmente, e estabelecidas a área total de vasos, o volume do órgão, a proporção de volume ocupada pelos vasos no volume total do órgão, área total do lúmen, do epitélio sensorial e não sensorial em ratos que já atingiram a maturidade. Apesar de não existir diferenças significativas no volume do órgão vomeronasal entre machos e fêmeas de ratos Wistar adultos, os componentes deste complexo órgão, apresentam notáveis diferenças. / The vomeronasal organ of 10 male and 10 female Wistar rats, adults, 120 days weighting between 210 and 318g, were used for this study to determine the histological structure and to verify the existence of sexual dimorphism. After perfusion, the organs were fixed by immersion in 2,5 glutaraldehyde in phosphate-buffered solution; isolated with the nasal septum, immersed in Perenyi solution for decalcification and then sectioned using classic morphometric and stereologic techniques. The sections qere placed on slides and stained with toluidina blue and analysed in light microscope. Diafanization of the organ was done and this sample was stained with acid hematoxilin. The constitution and distribution of the VNO tissue of male and female rats, sexually mature were described and quantified. We also established the vessel total area, total volume organ, the volume ratio occupied by the vessels in the total volume organ, total lumen area and total sensorial and no sensorial area. However there are no significative differences in the organ volume between male and female Wistar rats, the structures of this complex organ show remarkable differences.

ABSTRACT In humans, advanced malignancies are often targeted with broad-spectrum cytotoxic drugs that engender several detrimental side effects, in addition to their primary usage for eradicating cancerous cells. One of the lesser-researched of these effects, histological distortion of the olfactory system impedes a patient's ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from chemotherapy. Recent studies have indicated that cytotoxic drugs can damage gustatory epithelia immediately following administration (Mukherjee & Delay, 2011, 2013). We sought to observe the histological effects that cyclophosphamide (CYP), one of the oldest and most popular alkylating antineoplastic agents, may have on the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We utilized two immunohistochemical antibodies to label cells in the olfactory epithelia: anti-Ki67, a marker strictly associated with cell proliferation; and, anti-Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Twenty-eight C57BL/6 mice were administered a single intraperitoneal injection of CYP (75 mg/kg), while 20 control mice were administered saline, all at approximately seven weeks of age. Mice were euthanized at days one, two, six, 14, 30, and 45 post injection; subsequently, they were perfused with 4% paraformaldehyde, decalcified, cryoprotected, cryosectioned, and incubated with anti-Ki67 and anti-Keratin 5 antibodies, sequentially. Quantification results by fluorescent imaging of labeled sections revealed a significant decrease in the number of proliferative cells in the MOE and VNO of CYP-injected mice within the first 10 days post injection, followed by a compensatory period of increased cell proliferation through day 45 post injection, compared to saline-injected mice. Co-localization of horizontal basal cells and proliferative cells in the MOE and VNO of CYP-injected mice was significantly amplified at approximately 14 and 45 days post injection, respectively, compared to saline-injected mice. Our results suggest that administration of CYP can rapidly depress the populations of proliferative cells in the murine MOE and VNO; consequently, horizontal basal cells may afford restoration of the proliferative cell populations in the murine MOE and VNO, 14 to 45 days post injection, respectively.

Chez la souris, la puberté de la femelle est accélérée par des phéromones urinaires émises par le mâle (effet Vandenbergh). Les mécanismes neuroendocriniens sous-Jacents et les conséquences comportementales restent peu connus. Par une approche multidisciplinaire alliant immunohistochimie, chromatographie gazeuse couplée à la spectrométrie de masse et chirurgie expérimentale, mon travail de thèse montre que les neurones synthétisant la kisspeptine, un neuropeptide hypothalamique jouant un rôle essentiel dans le contrôle de la puberté, sont régulés positivement par les phéromones accélératrices de la puberté. Les neurones à kisspeptine reçoivent le signal phéromonal via le système olfactif accessoire et le transmettent aux neurones à GnRH. De plus, des analyses comportementales montrent qu’outre leur effet physiologique connu, les phéromones accélératrices de la puberté modifient à long terme le comportement sexuel de la souris femelle. En effet, la préférence de la femelle pour l’odeur du mâle s’exprime plus tôt à l’âge adulte après l’exposition péripubère aux phéromones émises par la souris mâle. / In the mouse, female puberty onset is accelerated by male urinary pheromones (Vandenbergh effect). The neuroendocrine mechanisms underlining this effect and the behavioral consequences are poorly understood. Through a multidisciplinary approach using immunohistochemistry, gas chromatography coupled to mass spectrometry and experimental surgery, my thesis research show that neurons that synthesize kisspeptin, a hypothalamic neuropeptide which plays a master role in the control of puberty onset, are positively regulated by puberty-Accelerating pheromones. Kisspeptin neurons receive pheromone signal via the accessory olfactory system and transmit it to GnRH neurons. Moreover, behavioral analyses show that besides their known physiological effect, puberty-Accelerating pheromones also have long-Term effects on sexual behavior of the female mouse. Indeed, puberty-Accelerating pheromones induce a precocious expression of male-Directed odor preference in adult female mice.