The pituitary-testicular axis in microgravity: analogies with the aging male syndrome.

Extraterrestrial exploration has gone on for decades before reversible testicular failure was shown to be a consequence of space flight in humans and animals at the end of the XXth century. This phenomenon was initially thought to depend on the psycho-physical stress expected to derive from a decidedly unusual environment, but the lack of consistent data concerning cortisol increase and/or gonadotrophin suppression pointed to the possibility of a primary defect. This was indirectly confirmed by the observation that a continuum of testicular androgen secretion potential exists from microgravity to centrifuge-derived hypergravity. Further experiments using tissue slices and suspended cells confirmed a direct inhibitory effect of microgravity upon testicular androgen production. A parallel deterioration of major physiological parameters, such as bone density, muscle mass/force, red blood cell mass, hydration and cardiopulmonary performance, has been repeatedly described during space missions, which, luckily enough, fully recover within days to weeks after landing, the time lag depending on single organ/system adaptation rates. According to the Authors of the present review, when taking together all reported changes occurring in space, a picture emerges closely resembling the so-called aging male syndrome, which is currently the object of daily screening and clinical care in their endocrine unit, so that microgravity may become a tool for better understanding subtle mechanisms of testicular senescence.

Morpho-functional alterations in testicular and nervous cells submitted to modelled microgravity.

Humans, as well as other life forms, have developed on earth under the terrestrial gravitational field. Questions concerning the effect of the gravity vector changes on the animal physiology have begun to emerge only in the last decades. Physiological alterations were observed during space flights, but space-born investigations at cellular levels are still very limited. Earth-bound simulations of low gravity obtained with the 3-dimensional Random Positioning Machine are extensively utilized to explore the effects of microgravity on cell function. After only a few minutes, weightlessness affected the cytoskeleton of lymphocytes, astrocytes, neurons and testicular cells, disorganizing microtubules, intermediate filaments and microfilaments. Cell division was impaired, mitochondria were disrupted and apoptotic phenomena occurred. Expression of proteins involved in transmembrane ion and water transport were also affected. In the Leydig cells the key enzymes (3beta- and 17beta-hydroxysteroid dehydrogenases) leading to testosterone synthesis were depressed. However, after 20 h of clinorotation the cells were able to synthesize heat shock proteins that initiated protection and recovery. The cytoskeleton was again well organized, normal mitosis occurred and the percentage of apoptotic cells returned to the range of 5%, similar to the control cultures. Ion and water transmembrane proteins and steroid dehydrogenases returned to normal levels. Long-term experiments showed that low gravity induced only transient alterations in the cultured cells, which were able to adapt to the gravity vector changes and to regain normal activity. These data may explain the physiological adaptation occurring in astronauts during and after space flights.

Microgravity-induced alterations in cultured testicular cells.

Cultured STe cells (2n karyotype) from swine testis were submitted to simulated microgravity using a 3D Random Positioning Machine for 5 min., 15 min., 30 min., 1 h and 23 h. Sample processing included: histological characterization of cell types, immunohistochemical identification of (i) microtubules (a-tubulin), (ii) alkaline phosphates, (iii) 3 beta-hydroxy-steroid-dehydrogenase (3?-HSDH), and histochemical lipid analyses. After 5 min. simulated microgravity a slight microtubule disorganisation occurred, which increased dramatically with increasing microgravity duration. After 23 h microtubule arrays were completely disrupted. 3 beta-HSDH immunostaining was detectable only in one cell type: under control conditions and 5 min. into microgravity immunoreactivity was strong, but completely disappeared thereafter. Immunostaining intensity for alkaline phosphates, a good marker for myoid cells, decreased after 15 min. in microgravity.

Ion transport proteins and aquaporin water channels in the kidney of amphibians from different habitats.

Amphibians are known to spend part of their life on land and return to water to reproduce. However, some urodeles spend their entire life in water, while others succeed in completely avoiding water even during reproduction. Osmoregulatory mechanisms must therefore be different in the diverse environmental conditions of their respective life histories. The architecture of the kidney is similar in all amphibians; as a consequence the ion-water equilibrium must be regulated in the different environmental conditions. We investigated the immunolocalisation of Na(+)/K(+)/Cl(-) cotransport proteins, sodium pump and water-channel proteins (aquaporins) in aquatic Amphiuma means means, Rana dalmatina, a species that returns to water to reproduce, and Speleomantes genei, a completely terrestrial species. The investigation was carried out with immunohistochemical methods using antibodies to Na(+)/K(+)/Cl(-) cotransport protein NKCC1 T4, Na(+)/K(+)ATPase alpha-subunit, water-channel aquaporin 3 and the inner mitochondrial membrane (AMA). Cotransport proteins and sodium pump, involved in ion reabsorption, are widely distributed in A. means and R. dalmatina and confined to the distal segment in S. genei; conversely water channels, involved in water reabsorption, are limited to the collecting duct in A. means and R. dalmatina and distributed in the proximal and collecting ducts in S. genei.

Microgravity-induced apoptosis in cultured glial cells.

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.

Microgravity-induced programmed cell death in astrocytes.

Cultured astrocytes were submitted to simulated microgravity using a Fokker clinostat under continuous rotation (60 rpm) for 15', 30', 1h, 20h and 32h. Samples processing included (i) nuclear stainings using Propidium Iodide and 4,6-diamidino-2-phenilindole, dihydro chloride, (ii) immunohistochemical identification of Caspase-7, (iii) identification of DNA fragmentation using the terminal dUTP nick end labelling and (iv) Scanning Electron Microscope analysis. After 30' at simulated microgravity the glial cells showed morphological evidence of apoptosis: cell shrinkage, chromatin condensation, nuclear blebs and fragmentation. The enzyme caspase-7 was present and DNA fragmentation was evident. After 32h the density of the cell population was much lower than that observed in controls.

Mitochondria-rich cells in gills and skin of an African lungfish, Protopterus annectens.

We used scanning electron microscopy, the vital dye DASPEI and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills and skin (opercular, dorsal and ventral) of the lungfish Protopterus annectens in its free-swimming conditions and at the beginning of aestivation. In the free-swimming period, the gills were short and thick and the pavement cells were extremely large (30-40 microns). The mitochondria-rich cells, which were distributed in the secondary and primary epithelium, occurred as two morphologically different types, i.e. elongated and oval, similar to the alpha and beta chloride cells of fresh water teleosts. In the skin, only one type of mitochondria-rich cells was found, resembling the alpha chloride cells. All the mitochondria-rich cells distributed in the gills and skin were labelled with anti Ca(2+)-ATPase serum indicating the possible uptake of Ca2+ at freshwater chloride cell level. At the start of aestivation, the skin and gills were covered by a thick layer of mucus and the epithelium of the gills was reduced. The mitochondria-rich cells were almost completely covered by the pavement cells.

Simulated microgravity induces alteration in the central nervous system.

To investigate whether the signs of neurophysiological impairment observed in flight may be traced back to cytomorphology, we undertook a ground-based study focusing upon the architecture of cultured glial cells under simulated microgravity obtained by three-dimensional clinorotation.

Somatostatin in the ovary of an African lungfish (Protopterus annectens): an in situ hybridisation, immunohistochemical, and autoradiographical study.

In mammals, somatostatin seems to be involved in the control of ovarian steroidogenesis. There have been no studies on the presence or actions of somatostatin in the ovary of nonmammalian vertebrates. The localisation of somatostatin-14 was examined immunohistochemically using the antibody to somatostatin-14 in the ovary of the African lungfish Protopterus annectens. Immunoreactivity was present in the granulosa cells of mature ovarian follicle examined by light microscopy. Using an oligonucleotide probe complementary to mRNA for somatostatin-14 and labelled at the 3'-end with alpha-35S, in situ hybridisation demonstrated somatostatin-14 mRNA distributed in cells showing the same localisation as that of the immunoreactive cells. Binding sites for SST-14 were identified with autoradiography using [125I]somatostatin-14. Binding sites were localised on granulosa and theca cells. Somatostatin-14 may be thus synthesised in the lungfish ovary.

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.

Somatostatin in lungfish kidney: an immunohistochemical, autoradiographical and in situ hybridisation study.

The localisation of somatostatin-14 (SST-14) was examined immunohistochemically using the antibody Ab-SST-14 in the kidney of the African lungfish Protopterus annectens. Immunoreactive cells were present in the proximal tubules. In situ hybridisation, using an oligonucleotide probe complementary to mRNA for SST-14 and labeled at the 3'-end with alpha-35S, showed SST-14 mRNA distributed in cells with the same localisation as seen for SST-14 immunoreactive cells. Binding sites for SST-14 were identified with autoradiography using 125I SST-14. Binding sites were concentrated on cells of the proximal tubules. It is suggested that SST-14 may be synthesised in the lungfish mesonephros.

Pro-opiomelanocortin (POMC) expression and immunolocalization of POMC-related peptides in the ovary of Protopterus annectens, an African lungfish.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha MSH) and beta-endorphin were used to localize pro-opiomelanocortin (POMC)-derived peptides in the ovary of the African lungfish Protopterus annectens by immunohistochemistry. Immunoreactivity was observed in the granulosa and the internal theca of the vitellogenic follicles. No immunoreactivity was observed in immature follicles. Using human POMC cDNA as the hybridization probe POMC-like mRNA was identified in situ in cells of the granulosa and internal theca of the vitellogenic follicles. No labeling was observed in primordial follicles. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of the opiate hormones.

Vasoactive peptides in the heart of Champsocephalus gunnari.

The presence of vasoactive peptides known to control cardiovascular functions in mammals and sub-mammalian vertebrates was investigated in the sub-Antarctic icefish Champsocephalus gunnari. Western immuno blotting was used to demonstrate immunoreactive atrial natriuretic peptide (ANP), angiotensin II (Ang II), bradykinin (BK) and endothelin-1 (ET-1) in heart homogenates. Immunohistochemistry was used to investigate the distribution of ANP, Ang II, BK and ET-1 in the cardiocytes of the three chambers of the heart (atrium, ventricle and the very short conus arteriosus).

Atrial natriuretic peptide, bradykinin, and angiotensin II-like immunoreactivity in the harderian gland of the terrapin Pseudemys scripta: response to osmotic stress.

The Harderian gland of the terrapin Pseudemys scripta has four types of acinar cells. Type IV cells are very similar to the salt secreting cells of the salt secretory glands of various marine vertebrates. The presence and localization of the Ile5-Angiotensin II, Atrial Natriuretic Peptide, and Bradykinin has been investigated by immunohistochemical methods. Immunoreactivity is confined to the type IV cells. Changes in the environmental salinity resulted in different patterns in the immunoreactivity especially after incubation with Ab-Angiotensin II and Ab-Atrial Natriuretic Peptide. Immunoreactive Angiotensin II cells are more numerous in animals maintained in distilled water, when reabsorption of sodium is needed. In contrast, immunoreactive Angiotensin II cells are very few in animals maintained in seawater. On the contrary, the number of immunoreactive cells for Atrial Natriuretic Peptide is high in seawater maintained animals, and weaker in animals in distilled water. The type IV cell may be considered a candidate for ion regulation in the terrapin Harderian gland.

The kallikrein-kinin and renin-angiotensin systems in the kidney of an African lungfish, Protopterus annectens.

Renin-like activity (RLA), angiotensin I converting enzyme-like (ACELA), and kallikrein-like activity (KLA), activities of the key enzymes of renin-angiotensin and kallikrein-kinin systems, were sought in the kidney of the African lungfish Protopterus annectens during the aquatic phase. RLA, examined by RIA (using porcine angiotensinogen as substrate), was 0.38 +/- 0.05 ng angiotensin I/mg protein/hr. ACELA and KLA were investigated in assays spectrophotometrically. ACELA, measured at 37 and at 20 degrees , was, respectively, 1.55 +/- 0.55 and 0.61 +/- 0.23 nmol hippurate/min/mg protein. KLA was 7.34 +/- 0.93 mU/mg protein in the crude kidney extract and 31.05 +/- 7.50 mU/mg protein after electrophoretic purification. Renal kininogenase activity was inhibited by 100% by D-Phe-Phe-Arg-chloromethyl ketone (10 microM), 98% by phenylmethylsulfonyl fluoride (2 nM), and 91% by aprotinin (1000 kIU). The apparent molecular weight of the renal kininogenase on SDS-PAGE was 27,000 Da. Both the renal enzyme and the purified glandular kallikrein, used as a control, have the same mobility on polyacrylamide gel electrophoresis. Immunoreactivities toward angiotensin II and bradykinin were localized by double immunostaining in the same cells of the proximal tubules. Putative angiotensin II receptors were demonstrated immunohistochemically, in the supranuclear region of proximal tubular cells, using an antibody to the sequence between amino acids 225 and 237 of the mammalian AT1 receptor.

Renin-like activity, angiotensin I-converting enzyme-like activity, and osmoregulatory peptides in the dogfish rectal gland.

Renin-like activity (RLA) and angiotensin I-converting enzyme-like activity (ACELA), two key enzymes of the renin-angiotensin cascade (RAS), were sought in the dogfish rectal gland. RLA was 1.1 +/- 0.2 ng Ang I/mg protein/hr after incubation with porcine angiotensinogen and 0.8 +/- 0.1 ng Ang I/mg protein/hr after incubation with homologous plasma. ACELA was 7.22 +/- 1.08 and 8.87 +/- 1.9 nmol hippurate generated/min/mg protein respectively, at 0 and 37 degrees. The presence of these enzymes may indicate the presence of an endogenous RAS-like system in the rectal gland. Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) binding sites were demonstrated autoradiographically in the subcapsular region of the gland, suggesting a possible interaction of the two hormones in the blind outer ends of the rectal gland tubules. Immunoreactivities toward Ang II, ANP, bombesin, vasoactive intestinal polypeptide (VIP), glucagon, and somatostatin were differentially localized in the rectal gland within three concentric zones with potentially different functional activities. In the capsule, there was a strong positive ir-glucagon reaction and a slightly weaker reaction for ir-somatostatin and VIP. In the blind outer ends of the tubules (in the subcapsular zone), strong immunoreactivity was present toward all the tested peptides except glucagon and somatostatin. In the inner zone and in the central canal, only a weak immunoreactivity toward Ang II and glucagon was observed.

Renin and angiotensin converting enzyme in elasmobranchs.

Renin-like activity (RLA) and angiotensin I converting enzyme-like activity (ACELA), the two key enzymes of the renin-angiotensin system (RAS), were sought in the elasmobranch Scyliorhinus canicula. Renal extracts were desalted in a G-25 and eluted in a G-100 Sephadex column (calibration 15,000-70,000). The fractions were concentrated in a vacuum device. A 48,000-MW fraction incubated with synthetic and porcine angiotensiongen generated angiotensin I estimated by RIA. This same fraction was vasopressor in rats and dogfish. ACELA was sought in gill, heart, liver, spleen, pancreas, intestine, kidney, gonads, brain, skin, and muscle of dogfish using a spectrophotometric assay. The highest level of ACELA was found in the gills followed by spleen, kidney, and brain (33.79 +/- 2.3, 29.56 +/- 1.0, 14.62 +/- 1.0, and 13.80 +/- 2.3 nmol hippurate/min/mg protein, respectively). Intestine, gonads, skin and muscle contained no measurable amounts of ACELA. Captopril inhibited enzymatic activity from all ACELA containing tissues.

Intrarenal localization of angiotensin II specific binding in rat fetuses.

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]-ileu-5-angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu-5-angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.

Angiotensin II vascular receptors in fetal and neonatal rats.

Specific binding sites for angiotensin II in aorta and renal arteries have been studied in rat fetuses (18th day of pregnancy) and 1-day-old newborn rats by binding studies in arterial membranes using [125I] ileu-5-angiotensin II. One type of angiotensin receptor was found both in fetuses and in the newborns; the capacity of this (RT) decreased immediately after birth (from 0.06 +/- 0.01 nM to 0.02 +/- 0.005 nM; +/- SEM) and the affinity (Kd) increased at birth (from 3.5 +/- 0.6 nM to 19.5 +/- 1.2 nM; +/- SEM). Localization of the specific binding sites was studied by autoradiography on arteries from fetal and newborn rats either perfused with iodinated angiotensin II by cannulation of the aorta or in vitro on cryostat sections incubated with the radioactive angiotensin II. Both in fetuses and in the newborn the binding sites were located in the tunica media of the arteries.

Angiotensin II specific receptors in subcommissural organ.

In the subcommissural organ of male rats, a circumventricular organ situated inside the blood-brain barrier, specific receptors for angiotensin II were demonstrated by binding studies on homogenated membranes and by autoradiography carried out on frozen sections using 125I-angiotensin II. The receptor sites were localized in the subnuclear region of cells of the subcommissural organ. A single class of binding sites was found whose capacity was modulated by changes in the sodium plasma concentration which led to variations in plasma volume.