New approaches concerning the testis of Astyanax lacustris (Characidae): immunohistochemical studies.

Astyanax lacustris, locally known as lambari-do-rabo-amarelo, is a study model for Neotropical fish. Testis of A. lacustris shows deep morphophysiological changes throughout the annual reproductive cycle. This work analyzed the distribution of claudin-1, actin, and cytokeratin as elements of the cytoskeleton in germinal epithelium and interstitium; the distribution of type I collagen, fibronectin, and laminin as extracellular matrix compounds; and the localization of androgen receptor in the testis of this species. Claudin-1, cytokeratin, and actin were present in the Sertoli cells and modified Sertoli cells, and actin was also detected in peritubular myoid cells. Type I collagen were in the interstitial tissue, laminin in the basement membrane of germinal epithelium and endothelium, but fibronectin was additionally detected in the germinal epithelium compartment. The labeling of androgen receptor was higher in peritubular myoid cells and undifferentiated spermatogonia, and weaker labeling was detected in type B spermatogonia. Therefore, the present work highlights new aspects of the biology of the testis of A. lacustris, and contribute to amplify the understanding of this organ.

Nonsteroidal anti-inflammatory drugs act as endocrine disruptors in Astyanax lacustris (Teleostei: Characidae) reproduction: An ex vivo approach.

Pharmaceutical products can act as endocrine-disrupting compounds (EDCs), affecting the physiological processes of animals, such as development or reproduction. This study aimed to investigate the influence of different concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac (DCF) and ibuprofen (IBU) alone and mixed (MIX) on gonadotropin gene expression and gonadal steroid release using Astyanax lacustris pituitary and testes explant systems, respectively. The explant organs were maintained for 12 h in Leibovitz (L-15) medium supplemented with 0, 0.1, 1, 10, 100, and 1000 ng L-1 of DCF, IBU, and MIX (ratio 1:1 of the same concentrations of DCF and IBU alone) and gonadotropin releasing-hormone (cGnRH2) stimulation in pituitary explants and human chorionic gonadotropin (hCG) stimulation in testes explants. The pituitary glands and the media from the testicular explants were collected for gene expression analysis including the ß subunit of the follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) and secreted gonadal steroid concentration analysis, respectively. Both DCF and IBU (alone and mixed) decreased pituitary gene expression of fshß and lhß and this inhibitory effect was evident even at low concentrations. In the testes, DCF and IBU did not change the levels of estradiol, and both pharmaceuticals increased the release of 11-ketotestosterone at low doses, while only IBU decreased the levels of testosterone in all concentrations. IBU's inhibitory effect in the testes was not triggered by the mixture of the two drugs. These results suggest that NSAIDs, may interfere in fish reproduction by acting as EDCs, thereby negatively affecting A. lacustris spermatogenesis and maturation.

Ovarian development and the reproductive profile of Astyanax altiparanae (Teleostei, Characidae) over one year: Applications in fish farming.

Astyanax altiparanae is a Brazilian species of substantial commercial, environmental and scientific importance; however, existing studies on its reproduction do not seem to provide enough details. In light of the increasing use of this species in fish farming and the need for basic studies for the development of new production technologies, we describe the structural and ultrastructural characteristics of the ovaries of A. altiparanae, and characterize the species' reproductive cycle. Females were collected monthly from March 2013 to February 2014, and reproductive management began in October 2013. The ovaries were removed, fixed in Karnovsky's fixative, and prepared for light microscopy, transmission electron microscopy and immunohistochemistry anti-PCNA. These techniques enabled us to characterize the ovaries, the germ cells, and the somatic cells in detail, as well as their changes over time. The reproductive cycle was characterized based on the monthly variation in the gonadosomatic ratio, the proportion of germ cells, and the rate of oogonium proliferation. The macroscopic analysis of the ovaries suggests that the vascularization pattern and color of the ovaries vary according to development. There are new types of analyses that can be applied even in the fish farming industry, such as a comparison between ovaries staining and weight or the frequency distribution of these colors throughout the year. This study also provides details on microscopic characteristics that have never before been reported for species of Astyanax, such as the presence of annulate lamellae in oogonia, the development of the zona pellucida from oocytes in the one-nucleolus step, and the development of the micropylar apparatus in oocytes in the cortical alveolar step. When the reproductive cycle was analyzed, this species was found to have a long period of spawning, with a reproductive peak from October to February and multiple spawning events, confirming the period already described in the literature. Variations in reproductive periods and the ability to reproduce in lentic environments suggest that A. altiparanae has the ability to respond quickly to environmental changes and exhibits high reproductive flexibility. All of these characteristics confirm the great potential of this species in the fish farming industry.

Characterization of undifferentiated spermatogonia and the spermatogonial niche in the lambari fish Astyanax altiparanae.

Undifferentiated type A spermatogonia are the foundation of fish spermatogenesis. This cell population includes the spermatogonial stem cell population, which is able to either self-renew or differentiate into cells that will generate the male gamete - the spermatozoa. Spermatogonia stem cells are located in a specific region of the testes known as the spermatogonial niche, which regulates spermatogonial stem cell function. This study characterizes undifferentiated type A spermatogonia and their S-phase label-retaining cell properties in Astyanax altiparanae testes. This is a fish species of substantial commercial, environmental, and academic importance. Two types of undifferentiated spermatogonia have been described in A. altiparanae testis: Aund* and Aund. Among the main differences observed, Aund* spermatogonia have an irregular nuclear envelope, decondensed chromatin, one or two nucleoli, and nuages in the cytoplasm; meanwhile, type Aund have a round nucleus. Aund* is preferentially distributed in areas neighboring the interstitial compartment, whereas Aund is located in the intertubular area. Finally, this study found that undifferentiated type A spermatogonia were able to retain BrdU over a long chase period, suggesting that these cells have a long cell cycle and potential stem cell candidates among them. Based on these findings, undifferentiated type A spermatogonia may be characterized as putative stem cells in A. altiparanae testis. This work will contribute to further studies on the stem cell biology of this promising Neotropical experimental model.

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.

Spermatogonia, Germline Cells, and Testicular Organization in the Characiform Prochilodus lineatus Studied Using Histological, Stereological, and Morphometric Approaches.

Prochilodus lineatus is an important representative of the order Characiformes and a species that offers great advantages to fish farming. Therefore, detailed knowledge of its reproductive biology can be applied to various fields of production and biotechnology. In this study, we have identified testicular germ cells during spermatogenesis and have evaluated the volumetric proportion of the testes occupied by structures of the tubular and intertubular compartments. In addition, the individual volume of type A spermatogonia was measured and used to estimate the mean number of these cells per testis. Gonads of adult P. lineatus males were extracted and fixed. Light and transmission electron microscopy were applied to fragments of three testicular regions. Histological, stereological, and morphometric analyses were performed. The stereological data suggest that components of the tubular and intertubular compartments of the P. lineatus testes present a uniform distribution in all three regions and therefore reflect regions with similar distributions of cell types. In addition, P. lineatus testes showed ∼0.6% of type A spermatogonia, as well as a predominance of cysts of primary spermatocytes and spermatids during the reproductive phase evaluated. The results from this study provide a better understanding of the morphology and structure of the testis and of the characterization of the type A spermatogonia in P. lineatus. The nuclear diameter of germ cells also decreases significantly during spermatogenesis. The data presented herein are the first of its kind for the order Characiformes and may be useful for future biotechnology studies on fish reproduction. Anat Rec, 300:589-599, 2017. © 2016 Wiley Periodicals, Inc.

Characterization of gonadotropic cells during continuous and seasonal spermatogenesis of two freshwater fish species: a histochemical and immunohistochemical study.

This work describes gonadotropic (GtH) cells and their morphological and immunohistochemical changes during the spermatogenic cycle of Serrasalmus maculatus (continuous spermatogenesis) and Pimelodus maculatus (seasonal spermatogenesis). GtH cells, widely distributed in the proximal pars distalis of the adenohypophysis, were characterized as round-shaped cells with eccentric nucleus, and cytoplasm with basophilic secretory granules and a variable number of vacuoles for both species. Immunohistochemistry against ß-follicle-stimulating hormone (Fsh) and ß-luteinizing hormone (Lh) in adjacent sections showed two separated GtH-producing cell populations, and a third population where both GtHs are expressed in the same cell for both species. In the seasonal spermatogenesis of P. maculatus, GtH cells seemed to be more abundant during developing and spawning capable phases. In contrast, no cyclic changes were detected in the continuous spermatogenesis of S. maculatus, except for the strong immunoreaction for Fsh and Lh in males with intense spermiogenesis. We conclude that changes reported here might reflect the type of spermatogenic cycle (seasonal or continuous) which are under different regulatory mechanisms (environmental and internal cues) controlling the reproduction in these species.

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 Energética de São Paulo, São 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.

Reprodução em cativeiro é um dos problemas enfrentados na aquicultura exigindo a manipulação de fatores ambientais e/ou tratamento hormonal. Assim, verificou-se experimentalmente as alterações gonadais presentes em indivíduos adultos de Astyanax altiparanae decorrentes da diminuição do nível da água. As coletas foram realizadas a cada quatro horas, iniciadas quatro horas antes e encerradas 28 horas após a estimulação, na Estação de Piscicultura da Companhia Energética de São Paulo, São Paulo, Brasil. As gônadas foram analisadas por microscopia de luz. As fêmeas apresentaram ovários na fase de apto à reprodução até 12h; em 16h, um estágio mais avançado desta fase; e, a partir das 20h, na fase de regressão. Os machos tiveram os testículos em fase de apto à reprodução até 8h; em 12h, um estágio mais avançado desta fase; e, a partir das 16h, o retorno à fase de apto à reprodução. A descrição morfológica foi corroborada pela proporção das classes celulares. As fêmeas apresentaram variação no índice gonadossomático, mas não foi estabelecido o esvaziamento das gônadas para nenhum dos sexos. O processo utilizado para a indução à reprodução foi considerado satisfatório, uma vez que ambos os sexos apresentaram redução no número de gametas maduros no final do período de amostragem.

Pituitary gland morphogenesis and ontogeny of adenohypophyseal cells of Salminus brasiliensis (Teleostei, Characiformes).

In this study, we describe for the first time the details of the pituitary gland morphogenesis and the ontogeny of adenohypophyseal cells of a South American Characiform species with great importance for Brazilian Aquaculture, Salminus brasiliensis (Characiformes, Characidae), from hatching to 25 days after hatching (dah), by histochemical and immunocytochemical methods. The pituitary placode was first detected at hatching (0 dah), and the pituitary anlage became more defined at 0.5 dah. The neurohypophysis (NH) development started at 3 dah, and the early formation of its stalk at 12.5 dah. An increase in adenohypophyseal and NH tissues was also observed, and in juveniles at 25 dah, the pituitary displayed similar morphology to that found in adults of this species, displaying the main features of the teleost pituitary. PRL cells were detected at 0.5 dah, together with ACTH and α-MSH cells, followed by GH and SL cells at 1.5 dah. ß-FSH cells were detected at 25 dah, while ß-LH cells at 5 dah. The pituitary development in this species comprises a dynamic process similar to other teleosts. Our findings in S. brasiliensis corroborate the heterogeneity in the ontogeny of adenohypophyseal cells in teleosts and suggest a role for adenohypophyseal hormones in the early development of this species.

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.

Morphologic study of the liver of lambari (Astyanax altiparanae) with emphasis on the distribution of cytokeratin.

Studies on the morphology of the liver of teleosts reflect some controversy in the interpretation of the data, but also provide confirmation of variations in the structure of the organ in several species. Thus, we intend to understand the specific structural organization of the liver of Astyanax altiparanae. Specimens were collected in the city of Andirá, Paraná, Brazil. The livers were processed according to histological routine for inclusion in Paraplast, and the sections were stained with HE and Mallory's trichrome or followed the protocol for fluorescence immunohistochemistry, anti-cytokeratin. The liver of A. altiparanae was covered by a capsule of connective tissue, without delimiting lobes. The hepatocytes had an arrangement in cords around sinusoids. Melanomacrophage centers were observed. The vascular components and intrahepatic pancreatic acini were distributed between hepatocytes. Presence of cytokeratin was detected in tissues that lined the liver and endothelial cells of sinusoids. The comparison of the liver of A. altiparanae to other characids corroborates with the fact that there is variation in the morphology of the liver even between closely related species. Moreover, it appears that in this species, endothelial cells of sinusoids can synthesize the cytokeratin filaments required for the regulation of blood flow in capillaries in adults.

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.

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.

Reproductive cycle of male matrinxa, Brycon cephalus

The matrinxa (Brycon cephalus) is an abundant and economically important fish found in the Amazon river basin in Brazil. The seasonal morphological changes in the testes of this fish were studied. Captive-bred specimens were obtained from a breeding center between March 1994 and February 1996. The testes were classified as being of the tubular, unrestricted spermatogonial type, in which four phases of germ cells (spermatogonia, spermatocytes (primary as secondary), spermatids and spermatozoa) were identified. Based on the macro, and microscopic analyses of testes, and on the gonadosomatic index, four stages of germ cell development (resting, maturation, mature and regression) were identified.