Maria Inês Borella

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Dehydroepiandrosterone increases β‐cell mass and improves the glucose‐induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose‐induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS‐1, IRS‐2, PI 3‐kinase, Akt‐1, and Akt‐2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA‐treated rats showed an increased β‐cell mass accompanied by increased Akt‐1 protein level but reduced IR, IRS‐1, and IRS‐2 protein levels and enhanced glucose‐stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes).

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli–germ and germ–germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli–germ and germ–germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.

Testes of Astyanax altiparanae: The Sertoli cell functions in a semicystic spermatogenesis

The Astyanax altiparanae (lambari) is a South American freshwater fish belonging to the family Characidae. Although some authors have described reproductive aspects of this species, this is the first study about the morphology of the testes throughout the annual reproductive cycle of A. altiparanae. Fish spermatogenesis differs from that in mammals as it occurs in cysts whose borders are defined by cytoplasmic processes of Sertoli cells, thus creating a favorable environment for spermatogenesis. The functions commonly attributed to fish Sertoli cells were investigated using stereological, light and electron microscopy in A. altiparanae. Results showed that when the Sertoli cells of A. altiparanae are in contact with germ cells, they plan a support function that culminates in the production of spermatozoa. After releasing spermatozoa, modified Sertoli cells form the duct epithelium, transform into secretory cells and release a secretion into the duct lumen where spermatids and sperm are located. Thus, the present study revealed important aspects of the testes of A. altiparanae, and propose a sequence of functions played by the Sertoli cells in this species.

Immunohistochemical study of pituitary cells in wild and captive Salminus hilarii (Characiformes: Characidae) females during the annual reproductive cycle

Freshwater fish that live exclusively in rivers are at particular risk from fragmentation of the aquatic system, mainly the species that migrate upriver for reproduction. That is the case of Salminus hilarii, an important migratory species currently classified as “almost threatened” in the São Paulo State (Brazil), facing water pollution, dam construction, riparian habitat destruction and environmental changes that are even more serious in this State. Additionally, this species show ovulation dysfunction in captivity. Our studies focused on the identification and distribution of the pituitary cell types in the adenohypophysis of S. hilarii females, including a morphometric analysis that compares pituitary cells from wild and captive broodstocks during the reproductive annual cycle. The morphology of adenohypophysial cells showed differences following the reproductive cycle and the environment. In general, optical density suggested a higher cellular activity during the previtellogenic (growth hormone) and vitellogenic (somatolactin) stages in both environments. Additionally, the nucleus/cell ratio analysis suggested that growth hormone and somatolactin cells were larger in wild than in captive females in most reproductive stages of the annual cycle. In contrast, prolactin hormone showed no variation throughout the reproductive cycle (in both environments). Morphometrical analyses related to reproduction of S. hilarii in different environmental conditions, suggest that somatolactin and growth hormone play an important role in reproduction in teleost and can be responsible for the regulation of associated processes that indirectly affect reproductive status.

Induced reproduction in a migratory teleost species by water level drawdown

Captive reproduction is one of the problems faced in aquaculture requiring the manipulation of environmental factors and/or hormonal treatment. Thus, we seek to verify experimentally which gonadal changes were present in mature individuals of Astyanax altiparanae arising from decreased water level. Collections were made every four hours, initiated four hours before and finished 28 hours after stimulation, at the Fish Farming Station of Companhia Energetica de Sao Paulo, Sao Paulo, Brazil. The gonads were analyzed by light microscopy. The females had ovaries in the spawning capable phase until 12h; in 16h, in a more advanced stage of spawning capable phase; and, from 20h, in the regressing phase. Males had testes in the spawning capable phase until 8h; in 12h, in a more advanced stage of spawning capable phase; and, from 16h, the return to the spawning capable phase. The morphological description was corroborated by the proportion of cell classes. Females presented variation on the gonadosomatic index, but it was not found an emptying of the gonad for neither sex. The process of inducing reproduction with water level drawdown was considered satisfactory, since both sexes presented a reduction in the number of mature gametes at the end of the sample period.

Toxicity of manganese metallodrugs toward Danio rerio

Manganese is an essential metal which can be neurotoxic in some instances. As Mn-based metallodrugs are ever more prevalent in clinical practice, concern regarding the toxic effects of Mn discharges to water bodies on the biota prompted us to study the physicochemical parameters of these complexes and to assess their acute toxicity toward adult Danio rerio individuals, particularly in terms of brain tissue damage. Our results show that the Mn(III)-salen acetate complex EUK108 is toxic, which can be rationalized in terms of its lipophilicity, stability and redox activity.

A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

BackgroundThe testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively.ResultsThe morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril.ConclusionsThe major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

Involution processes of follicular atresia and post-ovulatory complex in a characid fish ovary: a study of apoptosis and autophagy pathways

Recent studies seem to indicate that apoptosis and autophagy can act cooperatively in fish ovaries in order to achieve more effective ovarian regression after spawning. Considering the importance of tissue remodeling in ovarian functioning, we sought to morphologically characterize the involution processes of follicular atresia and post-ovulatory complexes using Astyanax altiparanae as an experimental model, and to determine the location of proteins involved in apoptosis and autophagy throughout this process. Fifteen females were collected after reproductive management. Fragments of the left ovaries were removed, fixed, and prepared for light microscopy and immunofluorescence analyses. The main characteristics of the involution processes were found to be consistent with previous descriptions. However, there were certain morphological peculiarities that do not appear to have been described for any other species thus far. These peculiarities may be related to the focus of this study on a single species, which allows for a more detailed investigation into morphological changes than studies on multiple species. Autophagy was also found to precede apoptosis in both involution processes in A. altiparanae. This may be related to the energy recycling process required before the removal of degenerated follicular cells by apoptosis. Thus, these results support the idea that there is crosstalk between autophagy and apoptosis pathways in ovarian involution processes, as well as the idea that the cell death pathways of these processes are conserved between teleost species with external fertilization.

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 β subunit (fshb), the luteinizing hormone β 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 β 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,20β-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-17β (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.