Comparative transcriptome analysis on four types of gonadal tissues of blotched snakehead (Channa maculata).

Blotched snakehead (Channa maculata) is an economically important freshwater fish in China, of which males grow much faster than females. To illuminate the molecular mechanism of sex differentiation and gonad development, RNA-Sequencing was performed to identify sex-related genes and pathway in gonads of 6-month-old normal XX females (XX-F), normal XY males (XY-M), XY sex reversal females (XY-F) and YY super-males (YY-M). The analysis showed that many differentially expressed genes (DEGs) had similar expression patterns in XY-F and XX-F, which were different from XY-M and YY-M. qRT-PCR indicated that Amh, Dmrt1, and Sox9 had relatively high expression in testes of XY-M and YY-M. Taking Amh as an example, there was a relative fold change of 1.0 in XX-F, 2.1 fold change in XY-F, 36.1 fold change in XY-M, and 26.0 fold change in YY-M. Cyp19a1a, Figla, and Foxl2 were highly expressive in ovaries of XX-F and XY-F. Taking Figla as an example, there was a relative fold change of 557 in XX-F, 304.5 fold change in XY-F, 5.6 fold change in XY-M, and 4.4 fold change in YY-M. KEGG analysis revealed many DEGs distributed in pathways related to sex differentiation, steroid hormone synthesis and growth, etc. Significant variation and trends in relative expression levels tested by qRT-PCR were consistent with those recorded by RNA-Sequencing. This is the first time that transcriptome of snakehead has been investigated systematically and in an integrated way. Large quantities of candidate genes involved in sex differentiation, gonad development and growth dimorphism were identified. The study provides useful resources for understanding sex differentiation and growth dimorphism, potentially assisting mono-sex production of snakehead in aquaculture.

Autophagy in nutrient storage cells of the Pacific oyster, Crassostrea gigas.

In oysters, nutrients are stored in a special type of cells referred to as vesicular-connective tissue cells (VCT-cells). These cells accumulate and provide nutrient to satisfy various needs of the organism, including gametogenesis. During the annual reproductive cycle, VCT-cells pass through a series of changes in their morphology associated with nutrients mobilization for developing germ cells. The results presented here show an approximately 33-35% increase in the number of autophagic vesicles in cytoplasm of VCT-cells in the gonadal area of C. gigas during the stage of active gametogenesis as compared to the resting stage of reproductive cycle. No destruction of VCT-cells due to autophagy or any other factors was observed, both in males and females. Our results indicate that autophagy does increase in VCT-cells of C. gigas and plays a certain role in nutrient mobilization from these cells.

Reproductive biology of the Neotropical catfish Iheringichthys labrosus (Siluriformes: Pimelodidae), with anatomical and morphometric analysis of gonadal tissues.

The reproduction of Iheringichthys labrosus (Lütken, 1874) from the Turvo River, Brazil, was studied using anatomical, biometric, histological, and ultrastructural techniques. Between April 2014 and March 2015, a total of 278 males and 512 females were captured bimonthly. The testes of Iheringichthys labrosus are fringed and possess a cranial spermatogenic region and an exclusively secretory caudal region. Histologically, the cranial region is composed of seminiferous tubules with spermatogenesis being completed in cysts. The spermatozoa are of the primitive type with a spherical head and have a rudimentary intermediate piece and a long tail with an axonemic arrangement of 9 + 2. The caudal region does not form an individualized gland, and cells in this testis area have characteristics of protein secretion. A variable density electron-dense secretion accumulates in the cisternae of the rough endoplasmic reticulum in the lumen of the seminiferous tubules and in the testicular ducts during maturation. The cortical alveoli are discontinuous, and the zona pellucida consists of three layers crossed by pore canals, and the follicular cells are squamous in the early stages of oogenesis and cuboidal in advanced stages. The gonadosomatic index was associated with the maturation of the gonads while the condition factor indicated that the fish feed less and utilize adipose reserves during the reproductive period. Males and females reproductively functional throughout the year with spawning being partial or multiple, similar to that reported in studies of the species in lentic environments.

Ultraestructura del ovocito maduro y en reabsorción de Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae) / Ultrastructure of the mature and in resorption oocytes of Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae)

Spondylus limbatus es una especie bajo protección especial en México, de la que existe poca información biológica y nada sobre estudios histológicos o de ultraestructura del ovario. El objetivo de esta investigación fue caracterizar la morfología ultraestructural de los gametos femeninos maduros y en degeneración. La gónada femenina de S. limbatus en estado de madurez presentó ovocitos postvitelogénicos de 60-70 µm de diámetro, que presentan el aspecto característico de células metabólicamente activas y altamente sintetizadoras. La membrana citoplasmática posee especializaciones destinadas a aumentar la superficie de absorción de la célula, las microvellosidades; el citoplasma presenta numerosos sistemas membranosos relacionados con la síntesis de material de reserva y secreción; y el patrón de organización nuclear altamente lobulado, y por consiguiente con una gran superficie que asegura el intercambio núcleo-citoplasma, se incorpora de forma estructural al proceso de vitelogénesis. Finalmente, se describen los cambios ultraestructurales resultantes de la lisis de los ovocitos: colapso de las membranas nuclear y citoplásmica, y presencia de células hemocíticas macrófagas.

Spondylus limbatus is a species under special protection in Mexico, of which there is little or no information in the literature of biological, histological or ultrastructural studies of the ovary. The objective of this research was to characterize the ultrastructural morphology of mature and degenerating female gametes. The female gonad of S. limbatus in mature state presented post-vitellogenic oocytes 60-70 µm in diameter, which have characteristics of metabolically active and highly synthesizing cells. The cytoplasmic membrane has specializations designed to increase the absorption surface of the cell, the microvilli; the cytoplasm presents numerous membranous systems related to synthesis of reserve and secretion material as well as the highly lobed nuclear organization pattern; a large surface that ensures core-cytoplasm exchange, is structurally incorporated into the vitellogenesis process. Finally, ultrastructural changes resulting from the lysis of the oocytes are described: collapse of nuclear and cytoplasmic membranes, and the presence of macrophage hemocytic cells.

Changes in ultrastructure of gonads and external morphology during aging in the parthenogenetic cladoceran Daphnia pulex.

The water flea Daphnia pulex, a small crustacean that lives in freshwater ponds, undergoes parthenogenesis and gamogenesis according to environmental conditions. In D. pulex, different morphological characteristics can be observed using a microscope during aging. In this study, we recorded the growth, reproduction, and survival indicators of parthenogenetic D. pulex females to analyze the morphological changes that occur during senescence. We observed the growth and development of the parthenogenetic females on the 7th, 14th, 21st, and 28th days by using scanning electron microscopy and ultrastructure of the gonads on the 7th, 14th and 21st days with transmission electron microscopy. We found that body length and reproductive capacity in the parthenogenetic individuals first increased and then decreased with aging, and the survival rate decreased gradually. The depression of the brood pouch on the back of the parthenogenetic females implied a decline in gonadal function and gradual aging. During senescence, the number of lipid droplets in the gonads of the parthenogenetic females decreased, and the integrity of the mitochondria and peroxisome was destroyed. The results of our study revealed the morphological characteristics of senescence in D. pulex.

[Transmission electron microscopic observation on gonad of Oncomelania hupensis offspring bred in Weishan Lake areas].

OBJECTIVE: To observe the morphological changes in the testes and ovaries of adult 12th-generation Oncomelania hupensis bred for 12 winters in Weishan Lake areas. METHODS: The offspring of the adult O. hupensis snails bred in the Weishan Lake that were originated from the Yangzhou section of the Yangtze River was defined as the experiment group, while uninfected, adult O. hupensis snails captured from the marshland of the Yangzhou section of the Yangtze River served as the control group. Snails were dissected and intact testicular and ovarian specimens were sampled, routinely fixed, dehydrated, embedded, polymerized in an oven and sliced on an ultramicrotome. The sections were visualized under a transmission electron microscope, and the ultrastructure of the snail gonad was compared between the experiment and control groups. RESULTS: Transmission electron microscopy showed "9 + 2" microtubules on the transverse sections of the tails of sperm cells in the testes of male snails in the control group, with triangular acrosomes and spiral, dense nuclei seen in the tip, while in the experiment group, the "9 + 2" microtubules disappeared on the transverse sections of the tails of sperm cells in the testes of male snails, with low chromatin density found in the tip. Transmission electron microscopy revealed clear nucleolus and nuclear membranes in the ova of female snail ovaries, and displayed yolk body, liposomes and endoplasmic reticulum in the cytoplasm, bilayer twists of nuclear membrane and a uniform nucleolus in the control group, while in the experiment group, smooth nuclear membrane and unclear nucleolus were observed in the ova of female snail ovaries, with few contents seen within cells. CONCLUSIONS: Following breeding for 12 winters in the Weishan Lake, the 12th-generation O. hupensis snails fail to fully adapt to the natural environment in northern China, and the remarkable changes in the ultrastructure of the snail gonad may be a cause of gradual decline and even extinction of O. hupensis in the Weishan Lake areas.

The Origin of a New Progenitor Stem Cell Group in Human Development.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

Spermatogenesis as a tool for staging gonad development in the gonochoric appendicularian Oikopleura dioica Fol 1872.

Oikopleura dioica, the only gonochoric species among appendicularians, has a spematozoon with a mid-piece and a conspicuous acrosome that, during fertilisation, undergoes a reaction forming an acrosomal process. To provide more insight into the spermatogenesis of a holoplanktonic tunicate species that completes its life cycle in three to five days, changes in the testis during individual growth have been examined. Spermatogenesis has been subdivided into seven stages based on ultrastructural features during the formation and organisation of the male gonad and the relationships between its macroscopic anatomy and the events of sperm differentiation. Gametes undergo highly synchronised differentiation due to the presence of widespread syncytial structures. Both meiosis and spermiogenesis are brief, and the passage from spermatocytes to spermatids involves a progressive segregation of the germ cells from the syncytial mass with the formation of large cytoplasmic bridges and volume reduction for nucleus compacting and cytoplasmic material changing. The nucleus is small and penetrated anteriorly by a complex acrosome and posteriorly by the distal centriole and part of the flagellum. In spermatids, the single, large mitochondrion appears laterally to the nucleus, and finally, in spermatozoa, it migrates into the mid-piece, wrapping the proximal portion of the axoneme. Because this mitochondrial position is reached only in the late phases of spermatogenesis, it suggests that appendicularians have derived oligopyrenic sperms in which the small nucleus results from adaptation to the assembly of numerous spermatozoa inside the narrow space of the testis compacted in the genital cavity. The formulation of a staging system of gonad development in a model tunicate species known for having the most compacted genome in chordates led to a comparison of histological observations with recent molecular data, improving the characterisation of its biology and life cycle in light of evolutionary implications.

Developmental asynchrony and antagonism of sex determination pathways in a lizard with temperature-induced sex reversal.

Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential gonads differentiate and shortly thereafter sex specific traits become dimorphic. However, this may not apply to squamates, a diverse vertebrate lineage comprising of many species with thermosensitive sexual development. Of the three species with data on the relative timing of gonad differentiation and genital dimorphism, the females of two (Niveoscincus ocellatus and Barisia imbricata) exhibit a phase of temporary pseudohermaphroditism or TPH (gonads have differentiated well before genital dimorphism). We report a third example of TPH in Pogona vitticeps, an agamid with temperature-induced male to female sex reversal. These findings suggest that for female squamates, genital and gonad development may not be closely synchronised, so that TPH may be common. We further observed a high frequency of ovotestes, a usually rare gonadal phenotype characterised by a mix of male and female structures, exclusively associated with temperature-induced sex reversal. We propose that ovotestes are evidence of a period of antagonism between male and female sex-determining pathways during sex reversal. Female sexual development in squamates is considerably more complex than has been appreciated, providing numerous avenues for future exploration of the genetic and hormonal cues that govern sexual development.

Effects of environmentally relevant sub-chronic atrazine concentrations on African clawed frog (Xenopus laevis) survival, growth and male gonad development.

Sub-chronic toxicity of environmentally relevant atrazine concentrations on exposed tadpoles and adult male African clawed frogs (Xenopus laevis) was evaluated in a quality controlled laboratory for 90 days. The aim of this study was to determine the effects of atrazine on the survival, growth and gonad development of African clawed frogs. After exposure of tadpoles to atrazine concentrations of 0 (control), 0.01, 200 and 500 µg L-1 in water, mortality rates of 0, 0, 3.3 and 70% respectively were recorded for the 90 day exposure period. Morphometry showed significantly reduced tadpole mass in the 500 µg L-1 atrazine exposed tadpoles (p < 0.05). Light microscopy on testes of adult frogs exposed to the same atrazine concentrations using hematoxylin and eosin (H&E) and Van Gieson staining techniques revealed gonadal atrophy, disruption of germ cell lines, seminiferous tubule structure damage and formation of extensive connective tissue around seminiferous tubules of frogs exposed to 200 µg L-1 and 500 µg L-1 atrazine concentrations. Ultrastructural analysis of the cellular organelles using transmission electron microscopy (TEM) revealed significant amounts of damaged mitochondria in testosterone producing Leydig cells as well as Sertoli cells. Biochemical analysis revealed reduced serum testosterone levels in adult frogs at all exposure levels as well as presence of six atrazine metabolites in frog serum and liver. The results indicate that atrazine concentrations greater than the calculated LC50 of 343.7 µg L-1 cause significant mortality in tadpoles, while concentrations ≥200 µg L-1 adversely affect reproductive health of adult frogs and development of tadpoles sub-chronically exposed to atrazine.

Regulating distal tip cell migration in space and time.

Gonad morphogenesis in the nematode C. elegans is guided by two leader cells, the distal tip cells (DTC). The DTCs migrate along a stereotyped path, executing two 90° turns before stopping at the midpoint of the animal. This migratory path determines the double-U shape of the adult gonad, therefore, the path taken by the DTCs can be inferred from the final shape of the organ. In this review, we focus on the mechanism by which the DTC executes the first 90° turn from the ventral to dorsal side of the animal, and how it finds its correct stopping place at the midpoint of the animal. We discuss the role of heterochronic genes in coordinating DTC migration with larval development, the role of feedback loops and miRNA regulation in phenotypic robustness, and the role of RNA binding proteins in the cessation of DTC migration.

Global DNA Methylation Changes in Nile Tilapia Gonads during High Temperature-Induced Masculinization.

In some fish species, high or low temperature can switch the sex determination mechanisms and induce fish sex reversal when the gonads are undifferentiated. During this high or low temperature-induced sex reversal, the expressions of many genes are altered. However, genome-wide DNA methylation changes in fish gonads after high or low temperature treatment are unclear. Herein, we compared the global DNA methylation changes in the gonads from control females (CF), control males (CM), high temperature-treated females (TF), and high temperature-induced males (IM) from the F8 family of Nile tilapia (Oreochromis niloticus) using methylated DNA immunoprecipitation sequencing. The DNA methylation level in CF was higher than that in CM for various chromosomes. Both females and males showed an increase in methylation levels on various chromosomes after high-temperature induction. We identified 64,438 (CF/CM), 63,437 (TF/IM), 98,675 (TF/CF), 235,270 (IM/CM) and 119,958 (IM/CF) differentially methylated regions (DMRs) in Nile tilapia gonads, representing approximately 0.70% (CF/CM), 0.69% (TF/IM), 1.07% (TF/CF), 2.56% (IM/CM), and 1.30% (IM/CF)of the length of the genome. A total of 89 and 65 genes that exhibited DMRs in their gene bodies and promoters were mapped to the Nile tilapia genome. Furthermore, more than half of the genes with DMRs in the gene body in CF/CM were also included in the IM/CM, TF/CF, TF/IM, and IM/CF groups. Additionally, many important pathways, including neuroactive ligand-receptor interaction, extracellular matrix-receptor interaction, and biosynthesis of unsaturated fatty acids were identified. This study provided an important foundation to investigate the molecular mechanism of high temperature-induced sex reversal in fish species.

Histological and histochemical features of the oogenesis in the simultaneous protandric hermaphrodite shrimp Exhippolysmata oplophoroides (Decapoda: Caridea).

This study analyzes the dynamics of the vitellogenesis process in the simultaneous protandric hermaphrodite shrimp Exhippolysmata oplophoroides, based on light microscopic observations. The ovotestes of the shrimps at the different gonadal development stages were removed, fixed and submitted an usual histological procedure (HE) and histochemical techniques (Bromophenol Blue, PAS/Alcian Blue, and Von Kossa tests). The germinative cells were classified into oogonias, and oocytes in stages I-IV based on the following features: cell size, cytoplasm appearance, presence of yolk granules, lipid droplets, chorion, and chromatin patterns. In the male initial phase of the gonadal development, the ovotestes present mainly oogonia and oocytes I and II while in the functional hermaphrodite phase, oocytes III and IV predominate in the peripheral zone of the gonads. Oocytes with an atypical appearance of the cellular components indicative of reabsorption were also observed. This study showed an increasing accumulation of proteins, carbohydrates and lipids occurring as the germ cells develop, being the yolk elements deposited in a sequence, in which proteins and carbohydrates are the first to appear both by an endogenous as well as also by an exogenous origin. The presence of calcium was detected mainly in oocytes I, II and inside those in reabsorption, being posteriorly mobilized to chorion constitution and/or to hemolymph due to its role during molting. Although the similarity of the germ cells shape among the crustaceans, this first histochemical characterization of E. oplophoproides ovary increases the comprehension of oogenesis in a caridean simultaneous protandric hermaphrodite species.

Gonadal Morphology and Gametogenesis in Japanese Red Coral Corallium japonicum (Octocorallia: Alcyonacea) Collected off Cape Ashizuri, Japan.

Colonies of the Japanese red coral Corallium japonicum Kishinouye, 1903 collected off Cape Ashizuri, Japan were gonochoric and produced gonads in siphonozooids annually, mainly during the spring season. Polyp anatomy, gonadal morphology and gametogenesis in this species were revealed by light and electron microscopy. A siphonozooid had a pharynx with a prominent siphonoglyph and eight mesenteries: two sulcal, two asulcal, and four lateral. A rudimentary retractor was found on one side of each mesoglea of these mesenteries. The retractor arrangement in the siphonozooid was reverse of what was described in the autozooids of octocorals. Gonads initiated as small protrusions on the mesenteries, except in the asulcal ones, and even at an incipient stage they were covered with a sac-shaped thin layer of mesoglea, which was continuous with the mesoglea of mesenteries. Gastrodermis enveloped the complete outer surface of the thin layer of mesoglea throughout gametogenesis in both oocytes and sperm cysts. Oocytes produced many microvilli on their cortical surfaces beneath the thin layer of mesoglea concomitantly with the accumulation of lipid globules in the cells, whereas in sperm cysts spermatocytes and spermatids increased in number without microvilli production, followed by synchronous spermiogenesis involving remarkable changes in the shape and position of organelles. Based on the comparison of patterns in gonadal development between octocorals including C. japonicum, hexacorals and scyphozoans, octocoral and stauromedusa species may be characterized by the fact that gametogenesis never occurs in the matrix of mesoglea, but rather exclusively within the thin sac of mesoglea surrounded by gastrodermis.

Seasonal gonadal development and age-related maturity patterns of introduced pumpkinseed (Lepomis gibbosus Linnaeus, 1758) in a heated thermal reservoir and an adjacent river reach.

Testis and ovarian maturation status, maturity profile and gonado-somatic index (GSI) were assessed in pumpkinseed (Lepomis gibbosus) collected from Mirgenbach, a cooling-water reservoir associated with a nuclear power plant, and from the River Moselle 7km downstream of the reservoir's thermal outflow. Histological investigation indicated that in both sexes, gonadal development of pumpkinseed in the heated reservoir was more advanced than in the cooler Moselle River throughout the breeding season. The histological maturity profile of reservoir males ranked by the advancement of sperm cells was highly correlated with its GSI (rs=0.73, P<0.001). GSI of females in the reservoir increased with the stage at maturity, but GSI was not significantly correlated with total length, age or growth rate of the individual. All sampled individuals of both sexes were mature at age 1 in the heated reservoir, whereas 48% of age 1 males and 57% of age 1 females were not mature in the river. GSI patterns suggest that males in the reservoir adopted one of two reproductive strategies (nesters or cuckolders), whereas no small males with large enough testes to be considered cuckolders were apparent in the river. The warm thermal regime of Mirgenbach Reservoir led to precocial maturity, early season reproduction, and the greater prevalence of apparent cuckolder males than would normally occur in this climatic zone.

Sex Chromosome Mosaicism in the Gonads of DSD Patients: A Karyotype/Phenotype Correlation.

Sex chromosome mosaicism results in a large clinical spectrum of disorders of sexual development (DSD). The percentage of 45,X cells in the developing gonad plays a major role in sex determination. However, few reports on the gonadal mosaic status have been published, and the phenotype is usually correlated with peripheral lymphocyte karyotypes, which makes the phenotype prediction imprecise. This study was conducted on 7 Egyptian DSD patients to demonstrate the effect of sex chromosome constitution of both blood lymphocytes and gonadal tissues on the phenotypic manifestations. Conventional cytogenetic and FISH analyses of blood lymphocytes were conducted, and laparoscopy with gonadal biopsy was performed for histopathologic examination and FISH analysis. Gonosomal mosaicism was detected in 3 patients who had a non-mosaic chromosome pattern in blood lymphocytes. Two patients showed the same type of sex chromosome mosaicism in both the blood and gonadal tissues but with different distributions. Two other patients revealed a non-mosaic pattern in both tissues. The present study elucidates the importance of examining sex chromosome mosaicism in gonadal tissues of DSD patients and highlights the critical role of 45,X mosaicism which can lead to serious effects during early gonadal organogenesis.

Exogenous estradiol alters gonadal growth and timing of temperature sex determination in gonads of sea turtle.

Temperature sex determining species offer a model for investigating how environmental cues become integrated to the regulation of patterning genes and growth, among bipotential gonads. Manipulation of steroid hormones has revealed the important role of aromatase in the regulation of the estrogen levels involved in temperature-dependent sex determination. Estradiol treatment counteracts the effect of male-promoting temperature, but the resulting ovarian developmental pattern differs from that manifested with the female-promoting temperature. Hypoplastic gonads have been reported among estradiol-treated turtles; however the estradiol effect on gonadal size has not been examined. Here we focused on the sea turtle Lepidochelys olivacea, which develops hypoplastic gonads with estradiol treatment. We studied the effect of estradiol on cell proliferation and on candidate genes involved in ovarian pattern. We found this effect is organ specific, causing a dramatic reduction in gonadal cell proliferation during the temperature-sensitive period. Although the incipient gonads resembled tiny ovaries, remodeling of the medullary cords and down-regulation of testicular factor Sox9 were considerably delayed. Contrastingly, with ovarian promoting temperature as a cue, exogenous estradiol induced the up-regulation of the ovary factor FoxL2, prior to the expression of aromatase. The strong expression of estrogen receptor alpha at the time of treatment suggests that it mediates estradiol effects. Overall results indicate that estradiol levels required for gonadal growth and to establish the female genetic network are delicately regulated by temperature.

Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines.

The histological and ultrastructural features of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus rapidly proliferating in Marikina River, Philippines were characterized during the fish's 2010-2011 reproductive season. The germinal compartment of the testes was composed of anastomosing tubules with cysts undergoing synchronous development. Spermatogenic cells were along the length of the testes indicate it to be of the unrestricted spermatogonial type. The spermatozoon is classified as type 1 ect aquasperm devoid of acrosome, has rounded nucleus, and a long flagellum - characteristics necessary for external fertilization. Male P. disjunctivus was reproductively active during half of the year-long study with peak spawning during the most rainy months (June-August) and prolonged recrudescence during the dry months (February-May). Results from this study form a histological baseline to describe the gonad dynamics and reproduction of this invasive fish species as well as provide possible means of mechanical control to curb the population of the fish in this river.

Ultrastructural changes and programmed cell death of trophocytes in the gonad of Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada, Eutardigrada, Isohypsibiidae).

The studies on the fates of the trophocytes, the apoptosis and autophagy in the gonad of Isohypsibius granulifer granulifer have been described using transmission electron microscope, light and fluorescent microscopes. The results presented here are the first that are connected with the cell death of nurse cells in the gonad of tardigrades. However, here we complete the results presented by Weglarska (1987). The reproductive system of I. g. granulifer contains a single sack-like hermaphroditic gonad and a single gonoduct. The gonad is composed of three parts: a germarium filled with proliferating germ cells (oogonia); a vitellarium that has clusters of female germ cells (the region of oocytes development); and a male part filled with male germ cells in which the sperm cells develop. The trophocytes (nurse cells) show distinct alterations during all of the stages of oogenesis: previtello-, vitello- and choriogenesis. During previtellogenesis the female germ cells situated in the vitellarium are connected by cytoplasmic bridges, and form clusters of cells. No ultrastructural differences appear among the germ cells in a cluster during this stage of oogenesis. In early vitellogenesis, the cells in each cluster start to grow and numerous organelles gradually accumulate in their cytoplasm. However, at the beginning of the middle of vitellogenesis, one cell in each cluster starts to grow in order to differentiate into oocyte, while the remaining cells are trophocytes. Eventually, the cytoplasmic bridges between the oocyte and trophocytes disappear. Autophagosomes also appear in the cytoplasm of nurse cells together with many degenerating organelles. The cytoplasm starts to shrink, which causes the degeneration of the cytoplasmic bridges between trophocytes. Apoptosis begins when the cytoplasm of these cells is full of autophagosomes/autolysosomes and causes their death.

Induced autoimmunity against gonadal proteins affects gonadal development in juvenile zebrafish.

A method to mitigate or possibly eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesized that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and, consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 immunization tests. Zebrafish (n = 329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized transcripts encoding targeted proteins by in situ hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthermore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential to develop auto-immunization-based technologies to mitigate fish reproduction before they reach maturation. However, the highly variable results between treatments and individuals suggest significant optimization should be performed to achieve the full potential of this technology.