David E. Kime

‘Classical’ and ‘non-classical’ reproductive steroids in fish

SummaryThe nature of the steroids secreted by gonads of fish differs significantly from those of the mammals. The ‘classical teleost steroids’, oestradiol, testosterone, 11-ketotestosterone and 17,20βP, have been measured in a wide variety of teleosts but do not always reflect biological activities. There is increasing evidence that teleost gonads may produce a number of ‘non-classical steroids’ which may play an important role in their reproductive biology. Such products include metabolites reduced at C5 to 5α or 5β, at C3 to 3α or 3β, or hydroxylated at 6-, 7-, or 21. Conjugation, as either glucuronides or sulphates, may contribute either to localization of action within the gonad or to production of pheromones during the immediate pre-spawning period. Although it is often possible only to measure the steroids for which assays are readily available, it is important to recognize that there may be production of steroids that are not detected by such assays, but which nevertheless play a major role in reproductive activity. In evolutionary terms, the teleost hydroxylases probably originated in the very early fishes and show parallels with their analogues in amphibians and mammals. Gene sequencing may reveal a possible common ancestor for all vertebrate 6, 7 and 11-hydroxylases.

Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio).

To determine whether early life exposure to tributyltin (TBT), an aromatase inhibitor, impaired reproductive function in fish, Danio rerio were exposed to environmentally realistic levels (0.01-100 ng l(-1)) of TBT from 0 to 30, 30 to 60, and 0 to 70 days post-hatch, and the sex ratio and sperm motility of the adults examined 3-5 months after cessation of exposure. Fish exposed for 70 days to 0.1 ng l(-1) of TBT, a concentration presently below the detection limit in water, showed a male biased population which produced a high incidence of sperm lacking flagella. At 1 ng l(-1), the motility of sperm was significantly lower than that of control fish, while at 10 ng l(-1), all sperm lacked flagella and, at 100 ng l(-1), milt volume had increased. The effect of exposure on sex ratio was similar after exposure from 0 to 70 and 0 to 30 days, but even 100 ng l(-1) gave only 65% males after exposure from 30 to 60 days. Effects on sperm motility and morphology and on milt volume were less pronounced after 30 day than 70 day exposure. Our data suggest that screening for aromatase inhibiting activity and assessment of its risks in early life to human and wildlife fertility needs to be urgently addressed, and that the reproductive toxicity of TBT may presently be underestimated.

The effects of pollution on reproduction in fish

Introduction Effects of pollution on the teleost ovary Ovarian morphology Lipid production and vitellogenesis Lipids Vitellogenesis Oocyte maturation, ovulation and spawning Egg numbers and viability Effects of pollution on the teleost testis Testicular morphology and spermatogenesis Sperm motility and survival Effects of pollution on the reproductive hormones Hypothalamic and pituitary hormones Ovarian steroids Testicular steroids Hepatic catabolism Other effects of pollution on reproduction Comments and future directions Conclusions Acknowledgement References page 52 53

Vitellogenesis as a biomarker of reproductive disruption by xenobiotics

There is increasing evidence that many xenobiotic chemicals can reduce the reproductive capacity of fish through disruption of the endocrine system. Hepatic stimulation of vitellogenesis in male and juvenile fish provides a sensitive indication of exposure to environmental oestrogens and oestrogenic chemicals. Moreover, perturbations of female vitellogenin levels can provide a useful marker of dysfunction caused by endocrine disruption at many sites in the reproductive axis. Vitellogenesis can therefore provide a useful indicator of endocrine disruption by xenobiotics. Both inhibition and stimulation of vitellogenesis can have direct repercussions on the reproductive capacity of fish. An important caveat in interpreting the results of such studies is that they must be related to other components in the reproductive system which may be more sensitive to disruption and have greater consequences for reproductive success.

Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals☆

Abstract Computer assisted sperm analysis (CASA) has been used to analyse the effects of cadmium and zinc on the motility of fish sperm treated either for 24 h after partial dilution in an extender, or during the final dilution step on the microscope stage during video recording. The progressive motility of catfish sperm, which was not affected by storage for 24 h, was decreased after exposure to 100 ppm cadmium or 2000 ppm zinc in extender for 24 h. Only 1000 ppm cadmium or 2000 ppm zinc had any effect on motility when added to sperm at the final dilution stage. These results suggest that concentrations of cadmium which have been found to be present in fish gonads as a result of bioaccumulation from the water or diet may decrease the motility of sperm during maturation or storage in the testis. Cadmium toxicity was significantly decreased in the presence of an equal amount of zinc, suggesting an equilibrium between zinc and cadmium at binding sites in the sperm. Computer assisted sperm analysis is shown for the first time to provide a rapid and quantitative measure of the effects of environmental pollutants on the quality of fish sperm. It is suggested that the methodology described may form the basis for a standard toxicity test to assess the potential hazards of environmental pollutants on reproduction.

Seasonal patterns of free and conjugated androgens in the brown trout Salmo trutta

Abstract A radioimmunoassay for the simultaneous determination of testosterone, 11β-hydroxytestosterone, 11-ketotestosterone, and their glucuronides is described. The plasma concentrations of these steroids have been determined throughout the annual reproductive cycle of the male brown trout Salmo trutta. The maximum concentration of testosterone (33.2 ± 5.4 ng/ml in late September) precedes that of 11-ketotestosterone (68.7 ± 5.5 ng/ml) which coincides with the period of spermiation in early November. Peak concentrations of testosterone glucuronide (14.5 ± 2.7 ng/ml), 11β-hydroxytestosterone glucuronide (6.8 ± 1.6 ng/ml), 11-ketotestosterone glucuronide (5.4 ± 0.7 ng/ml), and 11β-hydroxytestosterone (3.2 ± 0.7 ng/ml) were also found in November. The ratio of free:conjugated testosterone drops significantly between August and December in the male (10.7-0.66) but not in the female (6.8-5.3). Factors such as hormones and mineral ions which may affect glucuronyl transferase activity are discussed, and a possible role for glucuronides in the normal functioning of the reproductive cycle in the trout is suggested.

A strategy for assessing the effects of xenobiotics on fish reproduction.

Environmental pollutants, such as industrial and agricultural chemicals, heavy metals, drugs and products with hormonal activity may disrupt reproduction of aquatic wildlife such as fish. Such xenobiotics may cause disruption of the reproductive endocrine system, or they may directly affect gamete development and viability as a result either of their cytotoxicity or by altering the hormonal environment during gamete development. The various possible sites of action are reviewed, and it is suggested that tests for toxicity should isolate the specific component of the reproductive system that is most sensitive to such disruption and that this may be at levels well below that which causes mortality or visible signs of stress. Fish are proposed as the most suitable aquatic organism for such tests.

Gamete viability as an indicator of reproductive endocrine disruption in fish

Xenobiotic pollutants may disrupt reproductive endocrine function by acting at the hypothalamus, pituitary, gonad or liver. Disruption at any of these sites may result in either changes in the rate of gonadal development or in the viability of the gametes. Production of high quality gametes is dependent upon the correct hormonal milieu throughout their development, and any disruption of hormonal balance may result in abnormal sperm or eggs. Such effects may be apparent at levels of pollutant lower than that which causes arrest of gametogenesis or decreased gonadosomatic index. In this paper the use of sperm motility, morphology and fertilising ability in males, and egg numbers, size and hatching success in females as monitors of endocrine disruption will be discussed. Data are presented to show that exposure of adult zebra danios (Danio rerio) to 5 ng l−1 ethynylestradiol leads to arrest in development of the eggs produced at the early blastula stage and induces vitellogenesis in exposed males.

11-Oxygenated androgens in female teleosts: prevalence, abundance, and life history implications.

Although 11-ketotestosterone (11-KT) has been found in blood of females of several diadromous fish species, the importance, abundance, and prevalence of this and related 11-oxygenated androgens in females have not been investigated. To address this issue and to determine whether the differences among androgen profiles relate to specific life history strategies, particularly diadromous migrations, fish (males and females) of around 30 species were sampled and 5 androgens were measured by radioimmunoassay. Levels of 17beta-estradiol and cortisol were also determined to evaluate ovarian and interrenal activity at the time of sampling. Testosterone (T) was the predominant androgen in most sexually recrudescent females. Only in female eel and sturgeon were 11-oxygenated androgens present in levels as high as, or higher than, those of T, although substantial amounts were also found in blood of mullet and salmonids. 11-KT was generally the most abundant 11-oxyandrogen, levels being higher than those of 11beta-hydroxytestosterone or 11beta-hydroxyandrostenedione. It is concluded that 11-oxygenated androgens are quantitatively minor steroids in most female fish. There was no convincing evidence to support the notion that the presence of 11-oxygenated androgens in blood is an adaptation specific to migratory fishes.

An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss)

A simple and rapid Enzyme Linked ImmunoSorbant Assay (ELISA) is described and validated for testosterone, estradiol, and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). A general procedure for preparation of the acetylcholinesterase labeled steroid is described which is applicable to any steroid. Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 μl of plasma. The ELISA was applied to measurement of all three steroids every hour for over 24 hours in a female trout using cannulation of the dorsal aorta. This high sampling frequency revealed several short-term (<2 h) episodic pulses of testosterone and estradiol.