Histological study of the development and sex differentiation of the gonad in the European eel

The histological structure of the gonads was studied in yellow eels sampled from a coastal lagoon and from stocks reared in an aquaculture plant showing different sex ratios. Gonad development related to body size rather than to age and underwent an intermediate stage characterized by a structure of an early testis but containing oogonia and oocytes. This gonad was called the Syrski organ and the stage juvenile ambisexual. Ovaries were found in eels from 22–30 cm in length, possibly derived from undifferentiated gonads or from Syrski organs. Fully differentiated testes were found in eels >35 cm, derived from Syrski organs. These observations support the results of previous research. From elvers and in eels up to 15–16 cm in length, growth of the gonadal primordium is due to primordial germ cell migration. In eels > 15 cm multiplication of primordial cells begins. Oogonial clones were found in eels > 18 cm in length, whilespermatogonium B clones were observed in eels >30 cm in length. The dynamics of sex differentiation was different among stocks with different ultimate sex ratios: ovaries were found in shorter eels in stocks with a prevalence of females, in longer eels in stocks with a prevalence of males. This result supports the hypothesis of a metagametic (environmental) sex determination. The somatic cells in contact with germ cells and those in the interstitium appeared early during gonad development and preceded germ cell differentiation. This suggests that somatic cells are the targets of the environmental factors influencing sex differentiation.     

Sex Determination in Freshwater Eels and Management Options for Manipulation of Sex

Catadromous eels enter fresh water as sexually undifferentiated glass eels and develop into males and females before migrating back to sea as silver eels. Females develop ovaries directly from the ambiguous primordial gonad whereas males pass through a transitional intersexual stage before developing testes. Eels have sex-specific life-history strategies. Males may grow faster than females initially, but this difference is soon reversed and females attain a greater age- and size-at-metamorphosis than males. Male fitness is maximized by maturing at the smallest size that allows a successful spawning migration (a time-minimizing strategy) whereas females adopt a more flexible size-maximizing strategy that trades off pre-reproductive mortality against fecundity. Although heteromorphic sex chromosomes have been identified in some species, the sex of developing gonads is labile and gender is determined principally by environmental factors. Individuals experiencing rapid growth prior to gonad differentiation tend to develop as males, whereas eels that grow slowly initially are more likely to develop as females. Paradoxically, males tend to predominate under conditions of high density, which may be because a male “grow quickly, mature early” strategy increases an individual’s chances of survival during periods of intraspecific competition. High temperatures and saline conditions have also been proposed to favor development as males but experimental studies have failed to demonstrate a clear effect of either on sex determination. High proportions of female silver eels migrating from some upstream areas, lakes and large rivers may be due to low population density or poor conditions for growth in these habitats. Manipulating sex ratios in favor of females has the potential to increase eel production in aquaculture and to buffer natural populations against fishing pressure. Sex steroids (oestrogens and phytoestrogens) have a strong feminizing effect on undifferentiated individuals and are most effective when targeted at younger eels and administered at high doses for prolonged periods. Modifying local environmental conditions, in particular reducing eel density and limiting interference and social stress, may also promote the development of females. Further research into the timing and mechanisms of sex determination in eels is required to effectively and efficiently manipulate sex for conservation and/or economic benefit.     

Immunolocalization of steroidogenic enzymes in gonads of European eel, Anguilla anguilla (L.)

The localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P450arom) was investigated using polyclonal antibodies during gonad development in wild European eels, Anguilla anguilla (L.), from the River Po Delta (Ferrara, Italy). The first steroidogenic cells, observed in undifferentiated gonads of 14–16 cm yellow eels, showed no P450scc, 3β-HSD or P450arom activity, but positive regions appeared in head kidney insulae from this stage until the silver eel stage. In undifferentiated gonads of 16–20 cm yellow eels the steroidogenic cells were positive to all enzymes. Pre-Leydig steroidogenic cells, identified in Syrski organs of yellow eels of 22–26 cm evolving into testes, were positive to 3β-HSD and P450scc, but negative to P450arom. However, steroidogenic cells in Syrski organs evolving towards ovaries and in small but fully differentiated ovaries were positive to all enzymes. Immature testes of yellow and silver eels had Leydig cells positive to P450scc and 3β-HSD; the same reactions were also observed in some Sertoli cells of silver eel testes containing meiotic cells. Sex differentiation in A. anguilla apparently occurs through an initial female stage controlled by P450arom activity. Leydig and Sertoli cells appear involved in different steps of hormonal control of spermatogenesis: Leydig cells begin their steroidogenic activity before meiosis, while Sertoli cells begin their activity during meiosis.     

Decline in non-native freshwater eels in Japan: ecology and future perspectives

The occurrence, distribution, and biological characteristics of non-native freshwater eels were analyzed using 5524 eels collected from 16 sites in Japan between 1997 and 2005. Three hundred seventy-four fishes (6.8%) were identified as non-native European eels, Anguilla anguilla, while the remainder (93.2%) were native Japanese eels, A. japonica. The European eel was found at 7 sites (44%), including 3 rivers, 2 freshwater lakes, one brackish lake, and one sea bay, suggesting a wide rage of habitat use. This variability of habitat use was also evidenced by the otolith microchemistry, which showed that they had lived in not only freshwater but also in seawater habitats. The sites with European eel were localized within the vicinity of southern Japan where a number of these eels were cultivated in the early 1970’s, suggesting that some had escaped from the culture ponds or were released intentionally into nearby natural waters. The large body size (mean total length: 803 mm), pigmented skin, enlarged eyes, and relatively matured gonads (mean gonad somatic index: 1.9) found in non-native European eels indicated that most had metamorphosed into the migratory silver phase, suggesting their ability to initiate spawning migration. However, the proportion of European eels in Mikawa Bay in 1997 was more than 12%, which decreased markedly to less than 2% after 2001, corresponding to the recent decline in import of European glass eels for aquaculture. This suggests that the population of European eels will decrease in Japanese waters in the future.     

Gonad differentiation and body growth in Anguilla anguilla L.

Using histological sections, the gonads of samples of yellow and silver eels of two populations were examined. The populations were previously analysed for growth and sex ratio. The histological structures observed are similar to those described in previous publications for the European eel, Anguilla anguilla and to those indicated for the Pacific eel, A. japonica . Well differentiated gonads are present in the silver eels. In the yellow eels, ranging in age from 0 + to 2 + years and from a length of 20 cm to that at which they become silver, undif-ferentiated and both sex gonads are found. Histological evidence is presented which suggests that the ovary, found even in young and small eels, is completely differentiated at a very early stage. The testis-like gonad of the yellow eel is a more primitive, and possibly reversible, gonad which differentiates completely at the beginning of sexual maturation and the change to the silver phase.     

Effects of diet supplementation with carp ovary on gonad differentiation and growth of the European eel

Treating elvers of European eel Anguilla anguilla with mature carp ovary for 3–6 months during early growth induced female differentiation in 51·6–66·7% of treated animals compared with c . 5% in controls. The treatment also induced differentiation of ovaries in eels <13 cm L _T and a higher number of Syrski organs with ambisexual characters, and was most effective when administered at an early growth stage. The results could be attributed to the natural steroid content of the carp ovary. The total weight of treated animals at the end of the farm experiment was 84·7% higher than controls. The specific growth rate for weight was significantly higher in female yellow eels than in males, for both control and treated groups. The enhanced growth was related to induced feminization. A diet supplementation with mature carp ovary could be a good approach to control of sex differentiation and growth in eels.     

Karyological and gonadal sex of eels(Anguilla anguilla) from the German Bight and the lower River Elbe

Yellow eels(Anguilla anguilla) taken during summer from random commercial trapnet samples in the littoral area of Helgoland (n=116) and from a freshwater area of the River Elbe near Hamburg (n=109) were examined with regard to their karyological (i.e. existence of female sex chromosomes) and gonadal sex. In 47 % and 21 % of the two samples, respectively, chromosomes were unidentifiable because of insufficient numbers of mitotic plates. All eels from Helgoland, except one phenotypically undetermined fish, exhibited female gonads: 48 had female sex chromosomes and 13 were karyologically males. As found previously in the River Elbe, eels with male gonads predominated (n=55); 25 were undifferentiated. Of the gonadal males 26 were karyological males and 16 karyological females; the rest could not be identified by chromosome patterns. In contrast, all but one of the Elbe eels with female gonads (n=28) had female sex chromosomes. Some aspects of the sex reversal documented in the eel are considered.     

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.     

Effects of steroid sex hormones on chick embryo gonads in organ culture, with special reference to hormonal control of gonadal sex differentiation

At the initial stages of sex differentiation (7.5 and 8.5 days of incubation), chick embryo gonads were treated directly with testosterone or estradiol-17 beta in organ cultures. Chemically-defined media containing cholesterol as a steroid precursor were used. The differentiation of gonads in the 10 to 12-day controls, cultured in media containing no hormones, was close to that of gonads of equivalent age in ovo. Testosterone added to the medium exerted an inhibitory effect on the cortex of the female gonad and a masculinizing one on its medulla. The results of estradiol treatment confirmed the known feminizing effect of that hormone on the male gonad, the meiotic prophase in the genetically male germ cells being initiated in the induced cortex. These data may be interpreted in favour of a bihormonal theory of gonadal sex differentiation in birds, where the predominantly-synthesized male or female hormone in the gonad determines the male or female pattern of development of the corresponding gonad.     

Freshwater habitat use by a moray eel species,Gymnothorax polyuranodon,in Fiji shown by otolith microchemistry

Freshwater eels of the Anguillidae are diadromous because they migrate between ocean and freshwater environments, but other anguilliform fishes are generally considered to be strictly marine species. A few marine eels of the Muraenidae and Ophichthidae have occasionally been found in freshwater or estuaries, indicating that anguillids are not the only anguilliform eels that can use freshwater in some parts of the world. The moray eel Gymnothorax polyuranodon is one species that is known to be present in freshwater in the Indo-Pacific, but its life history is unknown. One way to evaluate what types of habitats are used by fishes is to determine the ratio of strontium (Sr) to calcium (Ca) in their otoliths, because this can show if they have used freshwater or saltwater environments. To evaluate the patterns of freshwater use by this unusual species of marine eel, the otolith Sr/Ca ratios of four G. polyuranodon (275–344 mm) caught in a freshwater stream of Fiji were analyzed. The consistently low Sr/Ca values (0–4) indicated upstream movement after settlement and freshwater or estuarine residence of all four individuals. These eels did not appear to have entered freshwater just for a short time period, which is consistent with other reports that this species is present in estuarine and freshwater habitats. This suggests that G. polyuranodon may be a catadromous species of marine eel. The similarities and differences between the life histories of anguillid eels and the few marine eels that have evolved the ability to invade freshwater habitats is discussed in relation to the evolutionary origin of diadromy in anguilliform fishes that originated in the marine environment.     

Cell proliferation is necessary for the determination of male fate in the gonad

Cell proliferation has been shown to have multiple functions in development and pattern formation, including roles in growth, morphogenesis, and gene expression. Previously, we determined that the earliest known morphological event downstream of the male sex determining gene, Sry, is the induction of proliferation. In this study, we used proliferation inhibitors to block cell division during early gonad development, at stages before the XY gonad has committed to the testis pathway. Using the expression of sex-specific genes and the formation of testis morphology as markers of testis determination, we found that proliferation within a specific 8-h window was critical for the establishment of the male pathway and the formation of the testis. Inhibition of proliferation before or after this critical period led to smaller gonads, but did not block testis formation. The critical period of proliferation coincides with the initiation of Sry expression and is essential for the differentiation of Sertoli cells, suggesting that proliferation is a vital component of the initiation of the male pathway by Sry. We believe these studies suggest that proliferation is involved not only in the elaboration of organ pattern, but also in the choice between patterns (male and female) in the bipotential gonad.     

Time lag of the response on the otolith strontium/calcium ratios of the Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis> to changes in strontium/calcium ratios of ambient water

We conducted a laboratory experiment to validate the relationship between the otolith strontium/calcium (Sr/Ca) ratio of Japanese eels (Anguilla japonica) and water Sr/Ca ratio when the ratio in water was changed. A linear and additive mixed modeling approach was used to assess otolith Sr/Ca ratio for elver-juvenile Japanese eels when ambient water was changed from seawater to freshwater. There was a significant difference between otolith Sr/Ca ratios of eels reared in freshwater and in seawater (freshwater: 1.3–2.3; seawater: 7.0–7.8 mmol/mol). The response of otolith Sr/Ca ratios of eels was not detected until after 10 d and models suggested that it might not be completed until at least 30–60 d. This study indicated the detailed ability of otolith Sr/Ca ratio to be used as a proxy for reconstructing the individual environmental history of Japanese eels. These findings can provide some assurances for future otolith Sr/Ca studies of eels in this system or in other areas that have similar environmental conditions.     

Sex differentiation in the European eel: histological analysis of the effects of sex steroids on the gonad

The effects of sex steroids on sex differentiation in the European eel were studied. The steroids, 17α-methyltestosterone (MT) and 17α-ethynylestradiol (EE), were given in the diet to 6–8 cm elvers and to 15–18 cm and 22–25 cm yellow eels. In our rearing conditions a very large percentage of the untreated eels developed as males. No masculinizing effect of MT could be demonstrated. The EE, administered at a dose of 10 mg kg^-1 of diet to 6–8 cm elvers and 15–18 cm eels, induced ovarian differentiation in about 90 and 65% of eels respectively, while in the control <5% of females was recorded. In 22–25 cm yellow eels a moderated feminizing effect was observed.
Histological analysis of the gonads of treated eels showed that sex steroids affect the gonadal structure. The androgen stimulates hypertrophy of compact connective tissue, early differentiation of Leydig cells, Sertoli cells and early formation of the spermatic duct. Oestrogen inhibits the differentiation of these structural components and stimulates the differentiation of follicular cells and an ovarian structure.
The involvement of gonadal structural components is discussed in relation to the effect of steroid treatment and to the peculiarities of sex differentiation in the European eel.     

Development and early differentiation of gonad in the european eel (Anguilla anguilla [L.], Anguilliformes,Teleostei): A cytological and ultrastructural study

The structure of the gonad of the European eel (Anguilla anguilla [L.]), an “undifferentiated” gonochoristic teleost, was investigated by transmission electron microscopy from 6–8 cm elvers to 22 cm yellow eels with juvenile hermaphroditic gonads. The pear-shaped gonads of 6–8 cm elvers assume, in 12–15 cm eels, a lamellar shape and enlarge by migration of germ cells, which we refer to as primary primordial germ cells. In the gonads of ∼ 16 cm eels, the primary primordial germ cells multiply, giving rise to clusters of germ cells that have ultrastructural characteristics of the primary primordial germ cells but show giant mitochondria, enlarged Golgi complexes, and round bodies not limited by membranes. We refer to these as secondary primordial germ cells. In 16–18 cm eels, syncytial clones of oogonia interconnected by cytoplasmic bridges are also observed. In 18–22-cm-long eels, the gonads contain primordial germ cells, oogonial clones, early oocyte cysts, single oocytes in early growth stages, and primary spermatogonia. Such germ cells are present in the same cross section where they are either intermingled or are in areas of predominantly female germ cells close to areas with predominantly male germ cells. These gonads are juvenile hermaphroditic and should be considered ambisexual because in larger eels they differentiate either into an ovary or into a testis. Somatic cells always envelop the germ cells following their migration into the gonad. These somatic cells first show similar ultrastructural features and then differentiate either into early Sertoli cells investing spermatogonia, or into early follicular (granulosa) cells investing the early previtellogenic oocytes. In eels ∼ 14 cm long, primitive steroid-producing cells also migrate into the gonad. In the ambisexual gonad they differentiate either into immature Leydig cells in the male areas, or into early special cells of the theca in the female areas. Nerve fibers are joined to the steroid-producing cells. Gonad development and differentiation are also associated with structural changes of the connective tissue characterized by the progressive appearance and deposition of collagen fibrils first in the mesogonadium, then in the gonad vascular region, and then in the germinal region. The collagen-rich areas are massive in the male areas and reduced in the female ones. J. Morphol. 231:195–216, 1997. © 1997 Wiley-Liss, Inc.     

Induced oogenesis of the European eel (Anguilla anguilla L.) in freshwater condition

European eel is a catadromous fish species, which means that after living in freshwater premature individuals adapt to sea water, and migrate to the Sargasso Sea for spawning. Although male eel can be sexually matured even in freshwater, to date, it was believed that female eel can be matured only in seawater. Here we show that the process of sexual maturation may be induced in freshwater by treating female eels with carp pituitary (GSI = 9.87 ± 1.55%). It is thus proposed that seawater condition is not an obligatory environment for stimulating gametogenesis and for artificial maturation of the European eel in neither gender.     

The structure of the gonad during natural sex reversal in Monopterus albus (Pisces: Teleostei)

A detailed study on the structure of the gonad of Monopterus albus was made as a basis for analysis of gonadal steroids in this sex-reversing teleost. Two types of males were identifiedand their existence appeared to be a result of the difference in gonadal ontogeny among the individuals in natural populations. The germinal area of the gonad, the gonadal lamellae, exhibited à zoned nature with regard to the location of the female and male germ cells. Observations suggested that the male germ cells originated from gonia pre-existing in the inner zone of the gonadal lamellae before sex reversal. Natural reversal of sex in this protogynous hermaphrodite was found to be usually a postnuptial event and was always accompanied by loss of ovarian tissue and by development of interstitial (Leydig) cells. In the mesogonial region of the gonadal wall, peculiar mesenchyme cells were found, their significance remained uncertain.     

The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage

The identification of five stages for female and two stages for male eels Anguilla anguilla using multivariate analysis was carried out on a large sample of individuals collected at six different locations in France. Stages corresponded to a growth phase (stages I and II), a pre‐migrant phase (III) and two migrating phases (IV and V). It is likely that an important period of growth triggered silvering through the production of growth hormone (GH) in stage III eels. In migrating eels gonad development, gonadotropin hormone (GTH‐II) production and increase of eye surface were similar at all sites. Differences among locations were found in gut regression and pectoral fin length. As variability for these increased with the size of the watershed and values were highest for the most downstream locations, fin length and gut regression may indicate the time since an eel started its migration.     

THE REPRODUCTIVE CYCLE AND SEXUALITY OF THE GREEN MUSSEL PERNA VIRIDIS (L.) (BIVALVIA: MYTILACEA) IN VICTORIA HARBOUR, HONG KONG

The reproductive cycle and sexuality of the green mussel Pernaviridis (L.) in Victoria Harbour, Hong Kong, were investigatedfrom July 1982 to May 1984. Histological studies showed thatthe cyde could be divided into 4 stages occurring in a highlyseasonal pattern. The very low (<0.1%) occurrence of functionalhermaphrodites indicates that P. viridis is gonochoristic. Pastreports of protandric rhythmic hermaphroditism may have resultedfrom reliance on fluctuations in sex ratio and sex identificationby gonad colour. These criteria are, however, considered invalidfor P. viridis because differences exist between the developmentalpatterns of the male and female gonads and, further, colourof the female gonad at the early proliferation stage resemblesthe male. Temperature was found to correlate positively withgonad development and with the lower threshold at 24°C.Temperature was not, however, limiting at some sites in HongKong. A joint Principal Component Analysis-Stepwise multipleregression procedure suggested that hypertrophication mightinhibit gonadal development in Victoria Harbour (Received 2 November 1987; accepted 30 December 1987)     

Colonisation of freshwater habitats by the European eel Anguilla anguilla

1. The spatial distribution of European eels in 18 U.K. rivers was related to distance from tidal limit using a negative exponential model. This function accounted for between 19 and 90% of the variation in eel density where quantitative data was available. For semiquantitative data the negative exponential function was a significant predictor of eel densities in only six out of 10 cases, although all rivers showed a consistent decline in abundance with distance upstream from the tidal limit. 2. The spatial distribution of different age groups of European eel in River Severn showed an initial rapid dispersion into freshwater followed by a much slower dispersion rate. Movement of the population upstream by a wave‐form migration process does not occur in this system. Instead colonisation of freshwaters can be seen as a two‐phase dispersion. Phase‐1 is a rapid dispersion upstream driven by density at the point source. Phase‐2 commences once the eels become yellow eels and is equivalent to random diffusion of particles. 3. These processes have important implications for the penetration of freshwaters with reduced numbers of eel larvae arriving on the coast of Europe and North America. Eel abundance will decrease more in freshwaters in an upstream direction whilst it may remain stable or decrease to a lesser extent in estuaries. They are also able to explain the demography of eels migrating upstream over weirs and the observations of varying sex ratios within catchments. We conclude that a dispersion model dependent on age, temperature, difficulty of migration, habitat quality and density of eels should be an important part of freshwater eel management.