Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.     

Morphological study for understanding the sexual pattern in ghost shrimp Callichirus major (Crustacea: Axiidea)

Callichirus major is a species of ghost shrimp that has burrowing habits, building underground galleries in the intertidal region of beaches. This study aimed to analyse some morphological parameters to verify its type of sexual pattern. A sample of 79 individuals, collected at the beach of Corujão, Espírito Santo, Brazil, was used. Data on the external morphology, presence of gonopores, carapace length (CL) and cheliped lengths (Lch) were obtained. In addition, the gonads were processed for histological analysis. The animals were divided into three categories: A, individuals with male characteristics; B, those presenting exclusively female characteristics; C, animals with characteristics of intersex, which presented supernumerary gonopores. Positive allometries were found for Lch versus CL only for category A, characterizing the group's marked heterochely. Histologically, it was possible to observe the presence of oocytes in category A and one case of male tissue in an intersex individual (category C). However, no genital ducts of both sexes were found in that individual. Callichirus major presents controversial sexual characters, which makes it difficult to understand the reproductive parameters and sexual pattern. However, maybe that species can develop a sexual system of hermaphroditism.     

Evidence for protandry in <Emphasis Type="Italic">Polydactylus quadrifilis</Emphasis> in the Kwanza Estuary,Angola, and its implications for local fisheries

A total of 141 Polydactylus quadrifilis were sampled from the Kwanza Estuary in Angola ranging in size from 436 to 1360 mm fork-length (FL). Of these, 124 were male, six intersex and 11 female. Female fish were significantly longer (mm, FL) and heavier (kg) than males and had significantly higher gonadosomatic indices (GSI’s) than those of males and intersex fish. Transitional (intersex) gonads were delimited, with testicular and ovarian regions separated by connective tissue. The first signs of ovarian tissue appeared on the outer ventral surface of the gonad. A second layer of ovarian tissue was first noticeable at either end of the initial ovarian region and developed back towards the centre of the ventral wall to form a luminal space. Early-stage oocytes were commonly found in the outer area of male regions and residual late-stage spermatids and spermatozoa were found in the luminal space of ovarian regions, suggesting a process of sex change from the outside inwards. A loss of male function was noted with increased ovarian prevalence. Based on this evidence it is suggested that P. quadrifilis in the Kwanza Estuary are protandrous. Owing to the reliance of P. quadrifilis on large highly fecund females for egg production, it is likely that they will be sensitive to fishing practices that target larger individuals within the population.     

A histological description of intersexuality in the roach

This paper is an illustrative guide to intersex in the roach Rutilus rutilus , based on 150 intersex individuals. Most intersex roach had female germ cells, or oocytes, within a predominantly male gonad (testis), and/or malformed/intersex reproductive ducts. The number, pattern and developmental stage of oocytes within testicular tissue in intersex fish varied greatly. In most intersex fish, a few primary oocytes, or numerous primary and secondary oocytes, were scattered randomly throughout the testicular tissue (multifocal intersex). In other, more severely feminized individuals, large areas of ovarian tissue were separated clearly from testicular tissue (focal intersex). Almost all intersex individuals had a female-like reproductive duct (ovarian cavity). In mild cases of intersex (in which the majority of the germ cells were male) the ovarian cavity was present together with the male sperm duct/vas deferens, whilst in certain severe cases, the sperm duct was absent or vestigial.     

Induction of gonadal intersex in genotypic male rainbow trout (Oncorhynchus mykiss) embryos following immersion in estradiol-17beta

This investigation was conducted to determine the initial period of gonadal sensitivity to estrogen in genetically male rainbow trout (Oncorhynchus mykiss). Fish were immersed in approximately 250 microg estradiol-17beta, (E2)/l for two 2 hr periods during different stages of embryonic development beginning 30 days postfertilization (DPF) and continuing until 68 DPF. Histological analysis of gonad samples indicated a significant proportion of E2-treated fish had intersex gonads; these gonads were primarily comprised of testicular tissue with one or more oocytes scattered throughout. The most sensitive period for altering normal testicular development was found to occur between 44 and 51 DPF (63% intersex), while the labile period in general was determined to span 24 days (from 30 to 54 DPF). Additionally, quantitative RT-PCR was used to measure estrogen receptor (ER) mRNA expression in whole individual untreated embryos at six weekly time points throughout the period of E2-exposure. Although the intersex condition was not observed throughout the entire E2-exposure period, ER mRNA was detected at each time point assayed.     

A histological study of maturity in male goldlined seabream Rhabdosargus sarba (Forskål, 1775) in the Oman Sea

The goldlined seabream Rhabdosargus sarba (Forskål, 1775) is farmed in several countries of the Western Pacific Rim and is a candidate species for aquaculture in the Gulf States of the Middle East. This study was undertaken to examine the onset and patterns of maturity in male goldlined seabream in support of the development of practical hatchery techniques. The testes of 337 male fish (0 + to 7 + , average FL 183 ± 20 to 315 ± 12 mm) collected from the Oman trap fishery (August 2001–April 2002; September 2002–2003) were classified into four stages, based on cell type (immature, developing, active or resting). Four spermatogenetic cell types were identified in the testes spermatogonia (types a and b), spermatocytes, spermatids and spermatozoa. Active testes appeared more regularly during the peak of the reproductive season in January and February. In March, resting testes were observed indicating the end of the reproductive season. There was clear evidence of protandry. Intersex gonads were observed in male fish from age 0 + to 7 + (33% at age 0 + to 75% at age 7 + with maximum 82% intersex frequency observed at age 5 + ). From the evidence presented, male fish age 1 + produced free‐flowing sperm under slight abdominal pressure during the period November–March.     

The histology of mature gonads of Oreochromis (Nyasalapia) karongae (Trewavas)

Histology of gonads of Oreochromis karongae was undertaken to study internal cell characteristics during maturation. This study was necessitated by low spawning output of the fish species. Several oocyte stages, ranging from primary forms to vitellogenesis, suggest that the maturation was generally succesfully attained in the fish ponds. Pre‐vitellogenesis oocytes (oogonia to perinuclear stage) and more advanced vitellogenesis (primary vesicle to tertiary yolk vesicle) oocyte stages were all found in the same gonads. However, there were some discontinuities observed during stages 3 and 4, suggesting selective maturation. Failure of gonads to mature normally is attributed to an ecological crunch that was in a previous study associated with environmental factors. Atretic oocytes were also recorded in the same gonads, a sign that some oocytes failed to mature normally. This indicates insufficient stimuli for normal gonad development. Several stages of spermatogenesis (spermatocytes, spermatid and spermatozoa) were also found in the same gonads. Selective recrudescence was more pronounced in O. karongae because generally less oocytes attained final maturation stages compared to Oreochromis niloticus and other tilapias. This could be the main reason for low natural breeding that has been observed in both wild and captive stocks, and led to the abandonment of its use in aquaculture. This study corroborates findings of previous studies that depended solely on external gonad characteristics. Histology provides conclusive evidence from internal cell characteristics that other techniques are unable to show.     

Steroid hormone profiles and correlative gonadal histological changes during natural sex reversal of sobaity kept in tanks and sea-cages

The gonadosomatic index of sobaity Sparidentex hasta fits well with the natural spawning season of February-March. At 12 and 24 months all the population matured as males. However, during this time, between April-September intersex gonads were seen. From October of the third year of life, females were seen for the first time. These changes in the histology of the gonads were correlated with the hormonal profile. Testosterone showed no significant correlation with sex but 11-ketotestosterone correlated very well with the mature male gonads and thus can be used for sexing. Oestradiol-17β higher in sex reversed individuals (females) in the spawning season than in males. The sequence of events in the protandrous sex change of this fish during the first two spawning seasons is male → intersex → male. However, during the third spawning season (age 36–37 months) this sequence is male →intersex → male or female (10–17%).     

Intersex Frogs Concentrated in Suburban and Urban Landscapes

The occurrence of intersex characteristics in amphibians has been linked to pesticide exposure in the laboratory and proximity to agricultural activity within natural populations. But, overall, the ecology of amphibian intersex is poorly known and, specifically, its occurrence in many landscape types and regions is unstudied. We offer the first analysis of the frequency of amphibian intersex across a range of land covers representing the major landscape types within a region. We used remotely sensed information to characterize land cover surrounding 4774 potential sampling locations within the Connecticut River Valley. From among these, we selected 24 ponds to collect postmetamorphic green frogs (Rana clamitans) from four land cover types: undeveloped, agricultural, suburban, and urban. Collected males were preserved and, then, prepared gonadal tissue samples were screened for the presence of testicular oocytes. A total of 233 animals was examined. Thirteen percent of all male green frogs had gonads containing testicular oocytes. Sexual abnormalities were not randomly distributed among sites or landscape types. Suburban landscapes had the highest frequency of abnormalities (21%), and both suburban and urban land covers were positively associated with the presence of abnormalities within a population. There was no evidence of a positive association with agricultural land cover. Examination of amphibian intersex across a range of contexts reveals that developed landscapes may be hotspots for abnormal sexual development. This new finding suggests that other mechanisms, not previously considered, could contribute to intersex in natural amphibian populations.     

Induction of gynogenesis and gonad development in the muskellunge

Muskellunge Esox masquinongy eggs activated with UV-irradiated yellow perch Perca flavescens sperm were subjected to heat shock at 31 ± 1° C for 6 min, 20 min after fertilization in three experiments. Survival at eyed stage was 1·7 ±1·6, 6·8 ± 4·8 and 2·3 ±0·5% in experiments I, II and III, respectively. After rearing the gynogenetic muskellunge in troughs and then in ponds, the sex ratio of gynogens in experiment I did not significantly (P>0·05) differ from the expected ratio 0:1 (male: female), however, one male and one intersex were observed. In experiments II and III, the sex ratio of gynogens differed significantly from expected (0 : 1). Three months after hatching, the growth of females did not significantly (P>0·05) differ among control and gynogenetic groups, whereas male growth was significantly (P<0·01) higher in the control v. gynogenetic group. The histological structures of the gynogenetic fish gonads in both sexes were similar to those described in the gonads of control muskellunge. After the overwintering period, signs of active spermatogenesis were observed in the testis of 1 + year old gynogenetic fish, whereas ovaries contained only oocytes at the perinucleolar stage. At this stage, plasma sex steroids testosterone (T) and oestradiol-17β (E2) cannot be used to discriminate the sex of gynogenetic muskellunge, as intersex, male and female fish had similar levels of T and E2.     

Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.     

Observations on the cytology of Bipes (Amphisbaenia) with special reference to its lampbrush chromosomes

The positions and general anatomical and histological characteristics of the gonads of Bipes biporus and B. canaliculatus are described. The amounts of DNA per haploid chromosome set have been measured in both species, the values being 1.83 and 2.0 pg for biporus and canaliculatus respectively. The karyotypes of both species are described on the basis of data from mitotic and meiotic metaphase chromosome sets and from lampbrush chromosomes. B. biporus has 10 macrochromosomes and 11 microchromosomes. B. canaliculatus has 11 macrochromosomes and 11 microchromosomes. The karyotypes of the two species differ distinctly with regard to the shapes of 3 of the macrochromosomes. Chiasma distribution is described for male meiosis in B. biporus. Studies of the lampbrush chromosomes of both species show the chiasma distribution in the female to be generally similar to that found in the male biporus. In B. canaliculatus, lampbrush chromosomes with maximally extended lateral loops are found in oocytes that are oblate spheroids measuring 0.7×1.0 mm along their short and long axes respectively, these being well before the start of the major phase of vitellogenesis. Smaller oocytes have more distinct chromomeres and shorter loops. Microchromosomes take the form of typical small lampbrush chromosomes in oocytes. There are at the most 1,000 chromomeres per haploid set of lampbrush chromosomes in B. canaliculatus. Chiasmata are described from lampbrush preparations in which the two half-bivalents are firmly attached to one another without evident association of their axes, indicating the possibility of chiasmate association between the DNA axes of lateral loops. There are remarkably few extrachromosomal nucleoli in Bipes oocytes, and its is suggested that this may indicate a level of ribosomal gene amplification that is much lower than that found in fish and Amphibia. The observations are particularly discussed in relation to current ideas concerning the structure and function of lampbrush chromosomes.     

High‐density aggregations of rodlet cells in the gonads of Greenland halibut Reinhardtius hippoglossoides,a deep‐water marine flatfish

Large aggregations of rodlet cells in the gonads of male and female Greenland halibut Reinhardtius hippoglossoides are reported for the first time. These rodlet cells were not arranged epithelially but rather were found throughout the connective tissue between oocytes (females) or within lymphatic spaces between testicular lobules (males). The reason for large aggregations of rodlet cells in the gonads and not other tissues of this species is uncertain.     

Early gonadal development and primary males in the protogynous epinepheline, Cephalopholis boenak

Early gonadal development of the protogynous epinepheline, Cephalopholis boenak, was examined histologically in 289 specimens with standard length (L_S) of 42–130 mm, collected from May 2000 to April 2002 in Hong Kong waters, to determine male developmental pathways and establish its sexual pattern. All juvenile gonads developed an ovarian lumen with primary‐growth stage oocytes and scattered spermatogenic tissue prior to sexual differentiation and first sexual maturation. From this bisexual phase containing both female and male tissues, some gonads differentiated as ovaries with further oocyte growth to cortical‐alveolus and vitellogenic stages, the rest differentiated as testes with the proliferation of spermatogenic tissue and the formation of a sperm sinus. All testes retained the lumen and primary‐growth stage oocytes, and sperm sinuses ran within the gonad wall. Unlike most protogynous species, among functional males it was impossible to distinguish those resulting from juveniles through sexual differentiation (i.e. primary male) from those resulting from functional females through sex change (i.e. secondary male) based solely on testicular morphology. A proportion‐spermatogenic‐tissue index (I_ST) was, therefore, developed and determined to be a reliable quantitative indicator for distinguishing differentiating, primary males before a sperm sinus was evident, from differentiating females during sexual differentiation. Since sexually transitional specimens with the concominant appearance of degenerating vitellogenic, or later, stage oocytes and spermatogenic tissue in the gonads were previously noted from Hong Kong, diandric, protogynous hermaphroditism is confirmed in C. boenak. For species, such as this and other epinephelines, in which all males have the same testicular morphology, a complete analysis of a wide range of body sizes from juveniles to adults is necessary for understanding male developmental pathways, and determining sexual pattern.     

Health assessment of freshwater fish species from Albasini Dam,outside a DDT-sprayed area in Limpopo province,South Africa: a preliminary study

During 2006 to 2008, the water, sediment and fish in Albasini Dam, used as a reference site outside a DDT-sprayed area, had been found to be as affected as those in the nearby DDT-sprayed area were. Accordingly, the aim of the current study, conducted in 2014, was to determine the health status of fish from Albasini Dam by means of a histology-based fish health assessment protocol. High levels of nitrates, as well as the organochlorine pesticides aldrin, dieldrin, chlordane and methoxychlor, were found in the water. Fish muscle tissue contained pesticides, including methoxychlor. A semi-quantitative histological assessment on Clarias gariepinus, Coptodon rendalli and Oreochromis mossambicus showed diverse histopathology in selected target organs, with the gills being most affected. Notably, the testis tissue of one C. rendalli was filled with eosinophilic liquid, characteristic of necrosis. Microscopic intersex was found in two of the nine O. mossambicus sampled. This is the second time that intersex as microscopic testicular oocytes has been reported in O. mossambicus in South Africa. This finding perhaps indicates that reproduction may be compromised and this will be investigated further. The presence of intersex and other histopathology in organs could be early warning signs of the declining water quality in this dam.     

Cloning and Characterization of a Rice Field Eel vasa-Like Gene cDNA and Its Expression in Gonads During Natural Sex Transformation

The vasa (vas)-related gene encodes an RNA helicase protein member of the DEAD-box family and plays key roles in germ-cell formation in higher metazoans. Using degenerate PCR and RACE, we cloned the vasa gene of the rice field eel (Monopterus albus), which is homologous to the Drosophila vasa gene. We named it ma-vas (Monopterus albus vas). Ma-vas encodes a protein of 618 amino acids, which contains all of the known characteristics of vasa homologs. RT-PCR analysis revealed that ma-vas was exclusively expressed in the gonads of the female, intersex, and male. During gonadal natural sex reversal, ma-vas is expressed in oocytes at all stages of oogenesis, in degenerating oocytes of ovotestis, and in spermatogonia and spermatocytes at early stages. The vasa positive signal was also observed in the peripheral layer of late ovary. It was not found, however, in that layer of the testis. Alkaline phosphatase (AKP) staining on the ovary and testis also indicated that some cells had differentiational potential in the peripheral layer of the ovary, suggesting that spermatogonia might arise from cells with AKP and vasa-positive staining in the peripheral layer of the female gonad.     

Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents.

Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all intersex and exposed male fish. In contrast to the effects observed in the intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in intersex roach. The estradiol-17beta concentration in intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in intersex fish compared with male fish. These data suggest a link between altered endocrine status in intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.     

The gonadal anatomy and sexual pattern of the protandrous sex-reversing fish, Rhabdosargus sarba (Teleostei: Sparidae)

The gonadal anatomy and sexual pattern of Rhabdosargus sarba (Teleostei: Sparidae) was studied to provide some basic structural information for the subsequent investigations on the endocrinology of protandrous hermaphroditism in this fish. Evidence derived from relating sex to differences in body size, from gonadal histology and from biopsy, revealed the occurrence of natural sex reversal from male to female in this species. The gonad of R. sarba possessed topographically distinct male and female zones that were well separated by connective tissue. Based on gross-anatomical and histological observations, four types of gonad were distinguished and were designated as Types I-IV in the present study. Active spermatogenetic tissue was present in the Type I, II and IV gonads. Dormant oogonia and perinucleolar oocytes were found in the Type I (male) and II (intersex) gonads, respectively. In the Type III (female) gonad, a functional ovarian zone was observed and the testicular tissue was vestigial. The existence of ovarian tissue as an oogonial band in the Type IV (male) gonad, which was found more commonly in large specimens, suggested that these functional males might not undergo sex reversal in their life cycle. The interrelationship of the different types of gonad is discussed with reference to protandrous hermaphroditism.     

The effect of inhibitors upon intracellular cyclic AMP levels and chick myoblast differentiation.

The small free-living nematode Caenorhabditis elegans is usually found as a hermaphrodite, but occasionally true males appear in the population. This study provides an account of gonadogenesis in the normal male and in a mutant that is a temperature-sensitive sex transformer.Male and hermaphrodite gonads develop from morphologically identical primordia. The small primordial gonad lies on the ventral side of the worm in the coelomic cavity. The gonadial primordium contains four nuclei at parturition. As this primordium develops in a hermaphrodite, it produces a double-armed, mirror symmetrical gonad that produces first sperm and then eggs. In the male, however, this primordium develops into an asymmetrical structure composed of a ventrally located testis, a loop region, a seminal vesicle, and a vas deferens. The male gonad presents a linear sequence of nuclei in successive stages of spermatogenesis beginning with a mitotic region in the testis, followed by clearly distinguishable stages of meiosis throughout the loop region to the seminal vesicle.A temperature-sensitive sex transformer mutant, tsB202, has been isolated. tsB202 carries an autosomal recessive mutation in linkage group II that at restrictive temperature transforms an XX hermaphrodite into a phenotypic male, complete with a normal male gonad and vestigial external genitalia. These transformed males are classified as pseudomales because they do not exhibit mating behavior. Temperature shift experiments have determined the specific temporal sequences of gonadogenesis, oogenesis, and spermatogenesis. Proper manipulation of the temperature regimen causes the production of intersexes. In one intersex, a male gonad complete with sperm, seminal vesicle, and vas deferens also contains oocytes. In another intersex produced by the complementary temperature shift, a hermaphrodite-shaped gonad develops that produces only sperm and no oocytes.