Gonadotropin subunits of the characiform Astyanax altiparanae: Molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity.

To better understand the endocrine control of reproduction in Characiformes and the reproductive dysfunctions that commonly occur in migratory fish of this order when kept in captivity, we chose Astyanax altiparanae, which has asynchronous ovarian development and multiple spawning events, as model species. From A. altiparanae pituitary total RNA, we cloned the full-length cDNAs coding for the follicle-stimulating hormone ß subunit (fshb), the luteinizing hormone ß subunit (lhb), and the common gonadotropin α subunit (gpha). All three sequences showed the highest degree of amino acid identity with other homologous sequences from Siluriformes and Cypriniformes. Real-time, quantitative PCR analysis showed that gpha, fshb and lhb mRNAs were restricted to the pituitary gland. In situ hybridization and immunofluorescence, using specific-developed and characterized polyclonal antibodies, revealed that both gonadotropin ß subunits mRNAs/proteins are expressed by distinct populations of gonadotropic cells in the proximal pars distalis. No marked variations for lhb transcripts levels were detected during the reproductive cycle, and 17α,20ß-dihydroxy-4-pregnen-3-one plasma levels were also constant, suggesting that the reproductive dysfunction seen in A. altiparanae females in captivity are probably due to a lack of increase of Lh synthesis during spawning season. In contrast, fshb transcripts changed significantly during the reproductive cycle, although estradiol-17ß (E2) levels remained constant during the experiment, possibly due to a differential regulation of E2 synthesis. Taken together, these data demonstrate the putative involvement of gonadotropin signaling on the impairment of the reproductive function in a migratory species when kept in captivity. Future experimental studies must be carried to clarify this hypothesis. All these data open the possibility for further basic and applied studies related to reproduction in this fish model.

Hypoxia inducible factor-dependent regulation of angiogenesis by nitro-fatty acids.

OBJECTIVE: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. METHODS AND RESULTS: The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 µmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. CONCLUSION: Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

Light-Induced photoreceptor degeneration in the mouse involves activation of the small GTPase Rac1.

PURPOSE: Rho GTPases play a central role in actin-based cytoskeleton reorganization, and they participate in signaling pathways that regulate gene transcription, cell cycle entry, and cell survival. This study verifies the role of Rac1 during light-induced retinal degeneration. METHODS: BALB/c mice were exposed to degenerative light stimulus, and their eyes were enucleated immediately or after the mice were kept in the dark for 6, 24, and 48 hours. Retinas were fixed and processed for immunohistochemical analysis. The distribution of Rac1 and its effectors-p21-activated kinases (PAKs) 1, 2, and 3-was studied by immunohistochemistry, whereas the expression of PAKs 3, 4, and 5 mRNA was analyzed by real-time PCR. Rac1 activity was measured using a pull-down assay. RESULTS: In control retinas, Rac1 was mostly observed in photoreceptors, plexiform layers, and Müller glial cells. In light-damaged retinas, some TUNEL-positive photoreceptors upregulated Rac1 expression. Conversely, most of the Rac1-positive cells were TUNEL-positive, mainly in early stages of retinal degeneration. The increase in Rac1 expression was preceded by enhanced Rac1 activity, detectable at the end of the light stimulus and still present 48 hours later. The distribution patterns of PAK1, PAK2, and PAK3 did not change in light-damaged retinas. However, there was a marked increase in PAK3 and PAK4 gene expression, whereas that of PAK5 mRNA remained the same. CONCLUSIONS: Rac1 may play a role in the apoptosis of light-damaged photoreceptors. The increased expression of PAK4 after light stimulus possibly functions as a protective mechanism against apoptosis.

The evolutionary origin of cardiac chambers.

Identification of cardiac mechanisms of retinoic acid (RA) signaling, description of homologous genetic circuits in Ciona intestinalis and consolidation of views on the secondary heart field have fundamental, but still unrecognized implications for vertebrate heart evolution. Utilizing concepts from evolution, development, zoology, and circulatory physiology, we evaluate the strengths of animal models and scenarios for the origin of vertebrate hearts. Analyzing chordates, lower and higher vertebrates, we propose a paradigm picturing vertebrate hearts as advanced circulatory pumps formed by segments, chambered or not, devoted to inflow or outflow. We suggest that chambers arose not as single units, but as components of a peristaltic pump divided by patterning events, contrasting with scenarios assuming that chambers developed one at a time. Recognizing RA signaling as a potential mechanism patterning cardiac segments, we propose to use it as a tool to scrutinize the phylogenetic origins of cardiac chambers within chordates. Finally, we integrate recent ideas on cardiac development such as the ballooning and secondary/anterior heart field paradigms, showing how inflow/outflow patterning may interact with developmental mechanisms suggested by these models.

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field.

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.