A new experimental model for the investigation of sequential hermaphroditism.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17ß-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

First Observation of a Spontaneously Matured Female European Eel (Anguilla anguilla).

This study reports on the first observation of a spontaneously matured female European eel. The 43-year-old eel, together with eleven other females, resided at an aquarium house since their capture in 2002 and stocking as glass eels in 1978. In June 2019, the girth of the belly of the female increased as a sign of oocyte maturation. The specimen had an estimated gonadosomatic index (GSI) of 47, only half of the oocytes were hydrated and matured, indicating that European eels are polycyclic batch spawners. The live eels of the cohort were still in the previtellogenic phase but their eye sizes were close to that of the matured eel. We hypothesize that substances released by other maturing and spawning fishes may have triggered puberty of the eel. This first observation, and the possibility of more eels maturing in the near future, provides a natural reference for the sexual maturation of the European eel.

A mechanistic model for studying the initiation of anguillid vitellogenesis by comparing the European eel (Anguilla anguilla) and the shortfinned eel (A. australis).

An inverse relation exists between the maturation stage at the start of the oceanic reproductive migration and the migration distance to the spawning grounds for the various eel species. The European eel Anguilla anguilla migrates up to 5-6000 km and leaves in a previtellogenic state. The shortfinned eel A. australis migrates 2-4000 km and leaves in an early vitellogenic state. In this study, we compared the early pubertal events in European silver eels with those in silver shortfinned eels to gain insights into the initiation of vitellogenesis. Immediately after being caught, yellow and silver eels of both species were measured and sampled for blood and tissues. Eye index (EI), gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated. Plasma 11-ketotestosterone (11-KT) and 17ß-estradiol (E2) levels were measured by radioimmunoassay. Pituitary, liver and ovaries were dissected for quantitative real-time PCR analyses (pituitary dopamine 2b receptor d2br, gonadotropin-releasing hormone receptors 1 and 2 gnrhr1 and gnrhr2, growth hormone gh and follicle-stimulating hormone-ß fshb; liver estrogen receptor 1 esr1; gonad follicle-stimulating hormone receptor fshr, androgen receptors α and ß ara and arb, vitellogenin receptor vtgr and P450 aromatase cyp19). Silver eels of both species showed a drop in pituitary gh expression, progressing gonadal development (GSI of ∼1.5 in European eels and ∼3.0 in shortfinned eels) and steroid level increases. In shortfinned eels, but not European eels, expression of fshb, gnrhr1 and gnrhr2, and d2br in the pituitary was up-regulated in the silver-stage as compared to yellow-stage females, as was expression of fshr, ara and arb in the ovaries. Expression of esr1 in European eels remained low while esr1 expression was up-regulated over 100-fold in silver shortfinned eels. The mechanistic model for anguillid vitellogenesis that we present suggests a first step that involves a drop in Gh and a second step that involves Fsh increase when switching in the life history trade-off from growth to reproduction. The drop in Gh is associated with gonadal development and plasma steroid increase but precedes brain-pituitary-gonad axis (BPG) activation. The Fsh increase marks BPG activation and increased sensitivity of the liver to estrogenic stimulation, but also an increase in D2br-mediated dopaminergic signaling to the pituitary.

Ovarian biopsy: a non-terminal method to determine reproductive status in giant kokopu, Galaxias argenteus (Gmelin 1789).

AIM: To establish a method of gonad biopsy for ovarian tissue collection in the declining giant kokopu Galaxias argenteus (Gmelin 1789) as an alternative to lethal sampling in order to understand the species' reproductive biology. METHODS: Six female giant kokopu weighing between 200 and 350 g were caught from the wild in early December of 2009 and transferred to a holding facility (Department of Zoology, University of Otago, Dunedin) where they were kept under a simulated natural photo-thermal regime for 10 months. Fish were repeatedly biopsied for ovarian tissue at near-monthly intervals (mean number of days between biopsies = 33) until ovulation. RESULTS AND CONCLUSIONS: Ovarian samples were successfully collected from giant kokopu by biopsy for use in downstream analyses. Among a total of 23 biopsy events, a single death occurred when a two-layered suturing approach was used, highlighting the value of this method for study of the reproductive biology of valuable fish. CLINICAL RELEVANCE: This biopsy method may have implications for veterinary research on fish physiology, pathology, conservation and development, when repeated tissue samples need to be collected over a prolonged period of time or for general surgical manipulations on fish when accessing the coelom. Furthermore, this approach allows the implementation of a more powerful experimental design, as repeated measures reduces the variability of estimates due to the removal of inherent stage differences among individuals.

Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis.

In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.

Ovarian mitochondrial cytochrome b mRNA levels increase with sexual maturity in freshwater eels (Anguilla spp.).

Differential display of mRNA was used to identify an upregulated gene in ovaries of artificially maturing Japanese eels (Anguilla japonica). Accordingly, mitochondrial (mt) cytochrome b, whose transcript levels increased from early to late vitellogenesis, was isolated, cloned and sequenced. Temporal trends in artificially maturing eels were compared with those in naturally and artificially maturing New Zealand eels (longfinned eel, Anguilla dieffenbachii; shortfinned eel, Anguilla australis) by Northern blot and in situ hybridization analysis to rule out any experimental artifacts. An increase in ovarian mt cytochrome b signals was seen when comparing immature and midvitellogenic longfinned eels, but not immature and early vitellogenic shortfinned eels, from the wild. Long-term captivity yielded reduced target mRNA levels, but abundance increased after hormonal induction of vitellogenesis. These results imply that the increase in mt cytochrome b mRNA levels during artificial maturation reflects natural development, although its onset appears to be brought forward during artificial maturation in the Japanese eel. It is suggested that increased mt cytochrome b mRNA levels result from both mitochondrial replication and increased transcription, and that they reflect the build-up of machinery for enhanced ATP-synthesis at some stage of oogenesis and/or early zygote development.

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis.

The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.

Changes in steroid hormone profiles and ovarian histology during salmon pituitary-induced vitellogenesis and ovulation in female New Zealand longfinned eels, Anguilla dieffenbachii gray.

To assess whether induced vitellogenesis in longfinned eels mimics that in naturally maturing conspecifics, female eels were artificially matured and steroid hormone status and oocyte cytology during oogenesis were evaluated. Successful induction of vitellogenesis was evident from the presence of yolk granules in the ooplasm of salmon pituitary homogenate (SPH)-injected, but not saline-, 17-hydroxyprogesterone-, and/or gonadotropin-releasing hormone-treated fish. In SPH-treated females, the migratory nucleus stage was reached after 33-53 days, followed by ovulation around 30 hours after induction of final maturation and ovulation. Only a portion of the germ cells matured, although resumption of vitellogenesis was seen in the majority of oocytes. In contrast, in ovaries of saline-injected controls, the most advanced oocytes were early vitellogenic. Atretic follicles were observed in ovaries of all eels, but abundance was greater in controls than in SPH-treated fish. SPH injections elevated plasma levels of estradiol-17beta and androgens, but not pregnenes, from within three days of treatment. Our results indicate that sex steroid levels in midvitellogenic hormone-treated females are similar to those in wild midvitellogenic females. In contrast, differences in yolk morphology of midvitellogenic follicles were seen between SPH-treated and wild females, especially in the second crop of midvitellogenic-sized oocytes measuring 300-400 microm in diameter. We discuss whether the observed differences affect egg quality, and perhaps explain the short life span of captive-bred eel larvae. J. Exp. Zool. 289:119-129, 2001.

A simple two-step method for the conversion of [3H]cortisol to [3H]-11-ketotestosterone.

Despite the existence of several protocols, problems appear to persist in the small scale chemical synthesis of radiolabeled 11-ketotestosterone from cortisol. We investigated the possibilities of using the mild oxidant pyridinium dichromate for the oxidative cleavage of the dihydroxyacetone side chain of cortisol and 17 beta-hydroxysteroid dehydrogenase for the subsequent reduction of the resulting 17-keto group. Our protocol has resulted in consistently high yields of both the intermediate, adrenosterone (70-80%), and the product, 11-ketotestosterone (up to 60%). This, taken together with the convenience and relatively low cost of our method, recommends the protocol for its use for the synthesis of [3H]-11-ketotestosterone for endocrine studies.