Franco Ricci

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis. In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains. In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules. Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

Identification and distribution of nitric oxide synthase in the brain of adult Antarctic teleosts

We investigated the presence of nitric oxide synthase (NOS) in brain of adult Antarctic teleosts by indirect immunofluorescence technique using a synthetic rat neuronal NOS (nNOS) antibody. The following species were examined: Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni (haemoglobin-rich), Chionodraco hamatus and Pagetopsis macropterus (haemoglobin-free). Immunoreactive cell bodies were localized in dorsal telencephalon, in hypothalamus, in optic tectum of the mesencephalon as well as in Purkinje cells of the cerebellum. No differences were observed in the localization of the nNOS immunopositivity in the Antarctic teleosts brains examined and NOS distribution was similar to that described in other teleosts, suggesting that nitric oxide (NO) may also function as a neurotransmitter in the brain of Antarctic teleosts. A strong immunopositivity was observed in the cerebral blood vessels of the icefishes suggesting that NO may play a pivotal role in the regulation of the cerebral blood flow especially in these haemoglobin-free species.

Ions and water transmembrane transport in nervous and testicular cultured cells in low gravity conditions

Aim of the present study was to investigate on the possible alterations induced by on ground modeled microgravity on ion-water transport proteins at cellular level. For the purpose we used astrocytes, C6 line, neurons (NT2 line from human teratocarcinoma) and testicular cells (germ cells, Sertoli cells, and Leydig cells; primary cultures from trypsinised prepuberal pig testes). Modeled microgravity was achieved by a desktop 3D Random Positioning Machine, cultures were kept rotating for 30′, 1h and 24h. After 30′, immunopositivity for the antibodies to Na+/K+ATPase and Na+/K+/Cl− co-transporters was greatly diminished, the plasma membrane appeared to be altered, and the mitochondria inner cristae were disrupted. Immunostaining to the antibody to the water channel aquaporin 4 was very bright. After 1h at random rotation immunostaining for the heat shock protein Hsp27 was visible, After 24h, immunostaining for the ion transport proteins was again like that of the controls, plasma membrane and the mitochondria were again normal. Immunostaining for aquaporin 4 become again similar to that of the controls. We conclude that low gravity induces only transient alterations in the cell’s transmembrane ion-water transport: the cells are able to adapt to the gravity vector changes in few hours.

Branchial morphology and ion–water transport proteins in Antarctic teleosts with different modes of life

In the Antarctic Ocean salt concentration differs from the bottom to the surface owing to the seasonal forming and melting of sea ice. Antarctic teleosts present different lifestyle from benthic to pelagic. While benthic animals face a constant seawater salinity, benthic–pelagic animals have to face different salt concentration. Branchial morphology and ion–water transport proteins were compared in animals with different lifestyle. The ultrastructure of the gills was investigated by scanning electron microscopy (SEM). Na+/K+/ATPase, Na+/K+/Cl− cotransport protein NKCC1 and Aquaporin 3 (AQP3), were investigated by immunohistochemistry. The immunoreactivity for the ion transporter proteins were more intense in the active benthic–pelagic animals and in the icefishes than in the sluggish benthic ones. Conversely, AQP immunoreactivity was stronger in the animals with sedentary lifestyles. The SEM showed the secondary lamellae in the benthic–pelagic animals more densely packed with the exception of the haemoglobin free teleosts.

Immunohistochemical localisation of FMRF-amide-like peptide in the brain of icefish and red-blooded Antarctic Teleosts

The distribution of Phe-Met-Arg-Phe-NH2 (FMRF-amide) -like immunoreactivity was investigated by indirect immunofluorescence technique using the molluscan FMRF-amide antibody in the brain of icefish (Pagetopsis macropterus and Chionodraco hamatus used as positive control) and red blooded (Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni) Antarctic Teleosts. Immunoreactive perikarya were localised in the ventral thalamus, in the hypothalamus (preoptic and periventricular regions) and in the intermedioventral rhombencephalon (vagal motor nucleus) as well as in the telencephalon and in the mesencephalon. Positive nerve fibres were seen to project towards the caudal brainstem to reach the rhombencephalon. No differences were observed in the immunopositivity of FMRF-amide new distribution in the Antarctic Teleosts examined. In the icefishes the immunoreaction was stronger than in the hemoglobin-rich Teleosts. The distribution patterns of the FMRF-amide immunostaining suggest that this peptide may play a pivotal role in the cardiovascular regulation in the Antarctic Teleosts.