Farnesol, a component of plant-derived honeybee-collected resins, shows JH-like effects in Apis mellifera workers.

Farnesol, a sesquiterpene found in all eukaryotes, precursor of juvenile hormone (JH) in insects, is involved in signalling, communication, and antimicrobial defence. Farnesol is a compound of floral volatiles, suggesting its importance in pollination and foraging behaviour. Farnesol is found in the resin of Baccharis dracunculifolia, from which honeybees elaborate the most worldwide marketable propolis. Bees use propolis to seal cracks in the walls, reinforce the wax combs, and as protection against bacteria and fungi. The introduction within a honeybee hive of a compound with potential hormonal activity can be a challenge to the colony survival, mainly because the transition from within-hive to outside activities of workers is controlled by JH. Here, we tested the hypothesis that exogenous farnesol alters the pacing of developing workers. The first assays showed that low doses of the JH precursor (0.1 and 0.01 µg) accelerate pharate-adult development, with high doses being toxic. The second assay was conducted in adult workers and demonstrated bees that received 0.2 µg farnesol showed more agitated behaviour than the control bees. If farnesol was used by corpora allata (CA) cells as a precursor of JH and this hormone was responsible for the observed behavioural alterations, these glands were expected to be larger after the treatment. Our results on CA measurements after 72 h of treatment showed bees that received farnesol had glands doubled in size compared to the control bees (p < 0.05). Additionally, we expected the expression of JH synthesis, JH degradation, and JH-response genes would be upregulated in the treated bees. Our results showed that indeed, the mean transcript levels of these genes were higher in the treated bees (significant for methyl farnesoate epoxidase and juvenile hormone esterase, p < 0.05). These results suggest farnesol is used in honeybees as a precursor of JH, leading to increasing JH titres, and thus modulating the pacing of workers development. This finding has behavioural and ecological implications, since alterations in the dynamics of the physiological changes associated to aging in young honeybees may significantly impact colony balance in nature.

Impact of captive conditions on female germinal epithelium of the butterflyfish Chaetodon striatus (Perciformes: Chaetodontidae).

Chaetodon striatus is a cosmopolitan seawater species present in aquaria all over the world and its extractivism is quite high. The lack of studies on the reproductive biology of C. striatus contributes to the difficulty in managing the species outside its natural habitat. Without knowledge of the mechanisms that control or affect gonadal changes, reproduction of C. striatus in captivity has become almost impossible, considering that the species is quite sensitive and the effect of captive conditions on its reproductive biology is unknown. Therefore, this study aimed to evaluate the effect on its reproductive biology of the animal's confinement and possible alteration in structure of the ovaries. In C. striatus, after oocyte development, for animals confined in small spaces, maturing oocytes undergo atresia. During atresia, ovarian follicles were at different stages of degeneration, characterized by the progressive loss of the basement membrane and disorganization of the follicle complex. In the advanced stage of follicular atresia, there was total loss of the basement membrane, culminating in degradation of the follicle complex. In unconfined animals, oocyte development and maturation were not affected. Confinement also affected the cell structure of the germinal epithelium, which showed large numbers of apoptotic bodies. The difference in cortisol and glucose levels between the unconfined and confined groups was significant, which may have to do with the change found in the ovaries, such as extensive follicular atresia and loss of the basement membrane.

In totum deparaffinization of biological samples and re-embedded in historesin for better diagnostic.

Paraffin has been the most traditional embedding medium used in histological techniques, however it can cause several technical artifacts. On the other hand, the use of plastic resins gives a more consistent and precise support to the tissues, allowing a better perfection of the histological sections. Thus, in an attempt to retrieve samples previously embedded in paraffin, we have described this new protocol which allows the material to be deparaffinized and re-embedded into historesin (glycol methacrylate). Paraffin embedded biological materials (en bloc) were deparaffinized and re-embedded with historesin. The histological sections of the samples (in paraffin and historesin) were analyzed under light microscope and the quality of the material was compared. As expected, samples embedded in historesin showed a better quality in their morphology, even if they were previously embedded in paraffin. Therefore, this new technique proposed here allows the recovery of old materials, through reprocessing to historesin, ensuring that materials already collected that cannot be obtained again are analyzed with greater clarity and great detail.

Presence of the matrix metalloproteinases during the migration of the primordial germ cells in zebrafish gonadal ridge.

In vertebrates, the primordial germ cells (PGCs) differentiate from extragonadal regions, migrating to gonadal ridge during the embryonic development. However, recent studies in mammals indicate that the PGCs originate from the epiblast and subsequently migrate into the yolk sac. Cell and molecular bases involved in routes during the migration of these cells are still not well understood. Thus, in an attempt to evaluate the participation of matrix metalloproteinases (MMPs) during the gonadal primordium formation in Danio rerio (zebrafish), the route of migration of PGCs was analyzed. In zebrafish, during the migration of the PGCs to the forming gonad, they bind by cytoplasmic processes to the extracellular matrix and migrate through amoeboid movements until they reach the gonadal ridge. During the epiboly, MMPs were not detected. However, after organogenesis, three MMP types were expressed in the somatic cells that were located ahead of the PGCs in the migration route. This expression was maintained throughout the mesentery and was not detected in the PGCs. Upon reaching the gonadal ridge, the PGCs and somatic cells express MMPs and epithelium begins to be formed. After the formation of the basement membrane, the germinal epithelium is delineated by the somatic cells, which remodeling the extracellular matrix. So, a PGC organization occurs through the tissue, forming the gonadal primordium. Concomitantly, granulocytes expressing different MMPs are present. This data in exposing the role of MMPs during the PGC migration to the forming gonad, may point a new way in understanding the reproductive biology of the vertebrates in general.

Cellular development of the germinal epithelium during the female and male gametogenesis of Chaetodon striatus (Perciformes: Chaetodontidae).

Butterflyfish Chaetodon striatus is highly sought after in the marine ornamental aquarium, although studies about its reproductive biology are scarce. Therefore, to contribute to a better understanding of the reproductive aspects of C. striatus, we describe in detail with the use of high resolution histology the cellular dynamics of the germinal epithelium during the reproductive life history of this species. Based on the activity of the germinal epithelium, this study describes different stages of the gonadal development, similar to the reproductive phases found in other fish, to determine the reproductive period of C. striatus. In characterization of gonadal development, the following germ cells are described for males: spermatogonia, spermatocytes, spermatids and spermatozoa. Oogonia, early, primary, secondary, full-grown and maturing oocytes are described for females. Female germinal epithelium of C. striatus showed substantial changes over the study period, indicating that there was an active spawning period. Male germinal epithelium also presented relevant alterations, indicating reproductive activity in the testicular lobules. Morphological data confirm how informative was the cellular dynamics of the germinal epithelium for understanding gonadal development during adult reproductive life of fish in general. Although Chaetodon are a popular species, previous studies have only produced superficial and rough histological analyses. Therefore, this study demonstrates important information on germinal epithelium of Chaetodon. This knowledge could be a fundamental tool for development of new strategies for breeding of several species in captivity, especially butterflyfishes.

Zygoparity in Characidae - the first case of internal fertilization in the teleost cohort Otomorpha

Most teleosts are externally fertilizing, with internal fertilization occurring as a relatively rare event. Until now, Euteleosteomorpha is the only teleost cohort known to undergo internal fertilization. In the teleost cohort Otomorpha, it has been recorded the presence of sperm in the ovaries of some species of Characiformes and Siluriformes, but no fertilized eggs have been found so far in the female reproductive tract. It has been presumed that oocytes can be released into the water with associated spermatozoa and only there becomes fertilized, and the term insemination has been used to characterize the strategy adopted by these fish. Here, we present the discovery of the first case of internal fertilization in the teleost cohort Otomorpha, in Compsura heterura (Characiformes: Characidae). In the course of spawning, the eggs form the perivitelline space and the animal and vegetative poles within the ovaries, evidencing oocyte fertilization. The newly spawned eggs then continue to form the animal and vegetative poles and increase the perivitelline space. These eggs are in the zygotic stage. These data indicate that fertilized eggs are only retained for a short period, providing evidence that C. heterura is a zygoparous fish.(AU)

A maioria dos teleósteos são espécies com fecundação externa, sendo a fecundação interna um evento relativamente raro. Até o momento, Euteleosteomorpha é a única coorte de teleósteos conhecida com espécies de fecundação interna. Na coorte de teleósteos Otomorpha, tem sido registrada a presença de esperma nos ovários de algumas espécies de Characiformes e Siluriformes, porém nenhum ovo fecundado foi encontrado até agora no trato reprodutor feminino. Presume-se que os oócitos possam ser liberados na água associados aos espermatozoides e que somente lá são fecundados, e o termo inseminação tem sido empregado para caracterizar a estratégia adotada por esses peixes. Apresentamos aqui a descoberta do primeiro caso de fecundação interna na coorte de teleósteos Otomorpha, em Compsura heterura (Characiformes: Characidae). Durante a desova, os ovos formam o espaço perivitelino e os polos animal e vegetal dentro dos ovários, evidenciando a fecundação interna. Os ovos recém-desovados continuam a formação dos polos animal e vegetal e aumentam o espaço perivitelino. Esses ovos estão na fase zigótica. Estes dados indicam que os ovos fertilizados são retidos por um curto período, fornecendo evidências de que C. heterura é um peixe zigóparo.(AU)

Presence, localization and morphology of TELOCYTES in developmental gonads of fishes.

Telocytes are a new defined type of interstitial cells, considered as a stem cell, with very long and thin cytoplasmic extensions. They are present in the vertebrates, and may participate in tissue remodeling. In fish, during gonadal development, the events that culminate with the germinal epithelium formation are well known. However, the interstitial compartment remains poorly explored, although it may have a great contribution to the morpho-functional changes that occur in the gonad. As in other organisms, in fish, the interstitium consists especially of connective tissue elements. However, until now, there are no reports of the presence and the action of the telocytes in the connective tissue of gonads of fish. Thus, this study aimed to detect the presence, localization and morphology of telocytes during the gonadal development of several species of fish. The gonads were analyzed by light microscopy, transmission electron microscopy and immunohistochemistry for localization of CD34, Vimentin, and metalloproteinases. The presence of two proteins characteristics of mesenchymal cell was detected in cells of the gonads of all species. In addition, they presented a typical morphology of telocytes, showing cellular extensions. Gonadal telocytes also presented positive response to metalloproteinases. In mammals, telocytes can undergo de-differentiation contributing to the reorganization of the extracellular matrix. This role may be performed by the metalloproteinases detected here. The detection of Vimentin and CD34 in the same cellular type, associated with its morphological characteristics, allows us to conclude that some interstitial cells in Teleostei are considered telocytes, identical to the ones already described in mammals and other vertebrates.

Action of the Metalloproteinases in Gonadal Remodeling during Sex Reversal in the Sequential Hermaphroditism of the Teleostei Fish Synbranchus marmoratus (Synbranchiformes: Synbranchidae).

Teleostei present great plasticity regarding sex change. During sex reversal, the whole gonad including the germinal epithelium undergoes significant changes, remodeling, and neoformation. However, there is no information on the changes that occur within the interstitial compartment. Considering the lack of information, especially on the role played by metalloproteinases (MMPs) in fish gonadal remodeling, the aim of this study was to evaluate the action of MMPs on gonads of sex reversed females of Synbranchus marmoratus, a fresh water protogynic diandric fish. Gonads were processed for light microscopy and blood samples were used for the determination of plasma sex steroid levels. During sex reversal, degeneration of the ovaries occurred and were gradually replaced by the germinal tissue of the male. The action of the MMPs induces significant changes in the interstitial compartment, allowing the reorganization of germinal epithelium. Leydig cells also showed an important role in female to male reversion. The gonadal transition coincides with changes in circulating sex steroid levels throughout sex reversion. The action of the MMPs, in the gonadal remodeling, especially on the basement membrane, is essential for the establishment of a new functional germinal epithelium.

Identification of the molecular sex-differentiation period in the Siberian sturgeon.

Sexual development prior to gonadal sex differentiation is regulated by various molecular mechanisms. In fish, a "molecular sex-differentiation period" has been identified in species for which sex can be ascertained prior to gonadal sex differentiation. The present study was designed to identify such a period in a species for which no genetic sex markers or monosex populations are available. Siberian sturgeons undergo a slow sex-differentiation process over several months, so gonad morphology and gene expression was tracked in fish from ages 3-27 months to identify the sex-differentiation period. The genes amh, sox9, and dmrt1 were selected as male gonad markers; cyp19a1a and foxl2a as female gonad markers; and cyp17a1 and ar as markers of steroid synthesis and steroid receptivity. Sex differentiation occurred at 8 months, and was preceded by a molecular sex-differentiation period at 3-4 months, at which time all of the genes except ar showed clear expression peaks. amh and sox9 expression seemed to be involved in male sexual development whereas dmrt1, a gene involved in testis development in metazoans, unexpectedly showed a pattern similar to those of the genes known to be involved in female gonadal sex differentiation (cyp19a1 and foxl2a). In conclusion, the timing of and gene candidates involved with molecular sex differentiation in the Siberian sturgeon were identified.

Cellular development of the germinal epithelium during the gametogenic cycle of the golden mussel Limnoperna fortunei (Bivalvia: Mytilidae).

The golden mussel Limnoperna fortunei is an invasive species that has quickly dispersed and colonized several potential different habitats distributed all over the world, causing environmental and economic impacts. Thus, in order to contribute to a better understanding of the reproductive aspects of L. fortunei, we described the cellular dynamic of the male and female germinal epithelium during the annual reproductive life history of this species, with the use of high resolution histology. An approximate of 1 200 specimens of L. fortunei were sampled periodically on the upper Paraná River floodplain (Brazil), from March 2010 to December 2012. Based on the activity of the germinal epithelium and consequent germ cell development, this study has resulted in the recognition of the following reproductive phases: Developing, Spawning Capable, Regressing and Regenerating. In the characterization of these phases, the following germ cells were described for males: spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. Cell nests, oogonia, early prophase oocytes, previtellogenic oocytes and vitellogenic oocytes (early vitellogenic oocytes, middle vitellogenic oocytes and full-grown oocytes) were described for females. The morphological data and reproductive parameters obtained, showed the value of the cellular dynamics of the germinal epithelium, for the understanding of the cyclic gonadal events during the adult reproductive life of the mollusk in general. These results on the gametogenesis of this invasive species may be a fundamental tool for the development of control strategies and programs implementation, to reduce their proliferation and impacts in natural local environments.

The basement membrane and the sex establishment in the juvenile hermaphroditism during gonadal differentiation of the Gymnocorymbus ternetzi (Teleostei: Characiformes: Characidae).

Although there are several studies on morphogenesis in Teleostei, until now there is no research describing the role of the basement membrane in the establishment of the germinal epithelium during gonadal differentiation in Characiformes. In attempt to study these events that result in the formation of ovarian and testicular structures, gonads of Gymnocorymbus ternetzi were prepared for light microscopy. During gonadal development in G. ternetzi, all individuals first developed ovarian tissue. The undifferentiated gonad was formed by somatic cells (SC) and primordial germ cells (PGCs). After successive mitosis, the PGCs became oogonia, which entered into meiosis originating oocytes. An interstitial tissue developed. In half of the individuals, presumptive female, prefollicle cells synthesized a basement membrane around oocyte forming a follicle. Along the ventral region of the ovary, the tissue invaginated to form the ovigerous lamellae, bordered by the germinal epithelium. Stroma developed and the follicle complexes were formed. The gonadal aromatase was detected in interstitial cells in the early steps of the gonadal differentiation in both sexes. In another half of the individuals, presumptive male, there was no synthesis of basement membrane. The interstitium was invaded by numerous granulocytes. Pre-Leydig cells proliferated. Apoptotic oocytes were observed and afterward degenerated. Spermatogonia appeared near the degenerating oocytes and associated to SCs, forming testicular tubules. Germinal epithelium developed and the basement membrane was synthesized. Concomitantly, there was decrease of the gonadal aromatase and increase in the 3ß-HSD enzyme expression. Thus, the testis was organized on an ovary previously developed, constituting an indirect gonochoristic differentiation.

Male gonadal differentiation and the paedomorphic evolution of the testis in Teleostei.

Testis differentiation from representatives of the Otophysi (Cyprinus carpio), Percomorpha (Amatitlania nigrofasciata), and Atherinomorpha (Poecilia reticulata) was comparatively described. In the undifferentiated gonad of C. carpio, the primordial germ cells (PGCs) are scattered throughout the gonads while in A. nigrofasciata and P. reticulata the PGCs are restricted to the ventral periphery. In the dorsal region of the developing gonads, with the exception of C. carpio, somatic cell rearrangements result in the differentiation of the sperm duct. Pre-Sertoli cells wrap around single spermatogonia forming cysts that proliferate forming acinar-clusters. In C. carpio and A. nigrofasciata, the cysts in each acinar-cluster move away from each other, creating a central lumen. In C. carpio, the acinar-clusters then fuse to each other forming tubules that become lined by the germinal epithelium. Subsequently, the tubules anastomose dorsally and create the sperm duct. In A. nigrofasciata, the acinar-clusters elongate, forming lobules that individually connect to the sperm duct. These are lined by the germinal epithelium. In P. reticulata, the spermatogonial cysts remain in the acinar-cluster organization. Subsequently, developing ducts connect each cluster to the sperm duct and lobules subsequently develop. In the differentiated testis of C. carpio and A. nigrofasciata, spermatogonia are distributed along the lengths of the anastomosing tubules or lobules, respectively. However, in P. reticulata, the spermatogonia remain restricted to the terminal end of the lobules. Considering testis ontogeny, the spermatogonial acinar-cluster is the adult characteristic of more derived taxa that approximate the early gonad developmental stages of the basal taxa.

Activity of the ovarian germinal epithelium in the freshwater catfish, Pimelodus maculatus (Teleostei: Ostariophysi: Siluriformes): germline cysts, follicle formation and oocyte development.

Distinct types of oogonia are found in the germinal epithelium that borders the ovarian lamellae of Pimelodus maculatus: A-undifferentiated, A-differentiated and B-oogonia. This is similar to the situation observed for spermatogonia in the vertebrate testis. The single A-undifferentiated oogonia divide by mitosis giving rise to A-groups of single differentiated oogonia, each enclosed by epithelial cells that are prefollicle cells. Subsequently, the single A-differentiated oogonia proliferate to generate B-oogonia that are interconnected by cytoplasmic bridges, hence, forming germline cysts. The prefollicle cells associated with them also divide. Within the germline cysts, B-oogonia enter meiosis becoming oocytes. Meiotic prophase and early folliculogenesis occur within the germline cysts. During folliculogenesis, prefollicle cells grow between the oocytes, encompassing and individualizing each of them. The intercellular bridges disappear, and the germline cysts are broken down. Next, a basement membrane begins to form around the nascent follicle, separating an oocyte and its associated prefollicle cells from the cell nest. Folliculogenesis is completed when the oocyte and the now follicle cells are totally encompassed by a basement membrane. Cells derived from the ovarian stroma encompass the newly-formed ovarian follicle, and become the theca, thereby completing the formation of the follicle complex. Follicle complexes remain attached to the germinal epithelium as they share a portion of basement membrane. This attachment site is where the oocyte is released during ovulation. The postovulatory follicle complex is continuous with the germinal epithelium as both are supported by a continuous basement membrane. The findings in P. maculatus reinforce the hypothesis that ovarian follicle formation represents a conserved process throughout vertebrate evolution.

Germline cysts and the formation of the germinal epithelium during the female gonadal morphogenesis in Cyprinus carpio (Teleostei: Ostariophysi: Cypriniformes).

The formation of both germline cysts and the germinal epithelium is described during the ovary development in Cyprinus carpio. As in the undifferentiated gonad of mammals, cords of PGCs become oogonia when they are surrounded by somatic cells. Ovarian differentiation is triggered when oogonia proliferate and enter meiosis, becoming oocytes. Proliferation of single oogonium results in clusters of interconnected oocytes, the germline cysts, that are encompassed by somatic prefollicle cells and form cell nests. Both PGCs and cell nests are delimited by a basement membrane. Ovarian follicles originate from the germline cysts, about the time of meiotic arrest, as prefollicle cells surround oocytes, individualizing them. They synthesize a basement membrane and an oocyte forms a follicle. With the formation of the stroma, unspecialized mesenchymal cells differentiate, and encompass each follicle, forming the theca. The follicle, basement membrane, and theca constitute the follicle complex. Along the ventral region of the differentiating ovary, the epithelium invaginates to form the ovigerous lamellae whose developing surface epithelium, the germinal epithelium, is composed of epithelial cells, germline cysts with oogonia, oocytes, and developing follicles. The germinal epithelium rests upon a basement membrane. The follicles complexes are connected to the germinal epithelium by a shared portion of basement membrane. In the differentiated ovary, germ cell proliferation in the epithelium forms nests in which there are the germline cysts. Germline cysts, groups of cells that form from a single founder cell and are joined by intercellular bridges, are conserved throughout the vertebrates, as is the germinal epithelium.