Immunohistochemical localization of CYP1A, vitellogenin and Zona radiata proteins in the liver of swordfish (Xiphias gladius L.) taken from the Mediterranean Sea, South Atlantic, South Western Indian and Central North Pacific Oceans.

Cytochrome P4501A (CYP1A) monoxygenase, vitellogenin (Vtg) and Zona radiata proteins (Zrp) are frequently used as biomarkers of fish exposure to organic contaminants. In this work, swordfish liver sections obtained from the Mediterranean Sea, the South African coasts (South Atlantic and South Western Indian Oceans) and the Central North Pacific Ocean were immunostained with antisera against CYP1A, Zrp, and Vtg. CYP1A induction was found in hepatocytes, epithelium of the biliary ductus and the endothelium of large blood vessels of fish from the Mediterranean Sea and South African waters, but not from the Pacific Ocean. Zrp and Vtg were immunolocalized in hepatocytes of male swordfish from the Mediterranean Sea and from South African waters. Plasma Dot-Blot analysis, performed in Mediterranean and Pacific specimens, revealed the presence of Zrp and Vtg in males from Mediterranean but not from Pacific. These results confirm previous findings about the potential exposure of Mediterranean swordfish to endocrine, disrupting chemicals and raise questions concerning the possible presence of xenobiotic contaminants off the Southern coasts of South Africa in both the South Atlantic and South Western Indian Oceans.

Histological and immunohistochemical investigation on ovarian development and plasma estradiol levels in the swordfish (Xiphias gladius L.).

The paper reports a histological and immunohistochemical description of oocyte growth and ultrastructural aspects of zona radiata (ZR) formation as well as the relationship between plasma estradiol-17beta, (E2) levels and ovarian development in swordfish (Xiphias gladius L.) from the Mediterranean Sea. Ovaries were inactive during March to mid April; maturation occurred during late April to June and spawning in June and July. Zona radiata formation starts, as Pas positive material, in oocytes at the lipid stage. In this stage a deposit of electrondense material between oolemma and follicular cells appears. In the cortical alveoli stage and through the early vitellogenic stage, the deposition of a moderately electrondense material occurred on the inner side of the ZR. Finally, in late vitellogenic oocytes a third layer, made of microfibrillar material, appeared. The immunohistochemical analyses revealed that the initial internalisation of hepatic zona radiata proteins (Zrp) in the swordfish oocyte starts before the uptake of vitellogenin (Vtg) and that it is associated with the low previtellogenic E2 plasma levels, while a significant E2 increase in plasma is associated with the beginning of Vtg uptake. This would appear to confirm the hypothesis that the differential and sequential induction of zonagenesis and vitellogenesis may reflect a general feature of teleost oogenesis.

In vitro metabolism of pregnenolone to 7alpha-hydroxypregnenolone by rainbow trout embryos.

Tissues taken from rainbow trout embryos at several developmental stages, were incubated in the presence of radioactively-labelled pregnenolone in order to determine the capability of salmonid embryos to metabolize steroids, such as pregnenolone, that are incorporated into the oocyte during gonadal growth and maturation. High performance liquid chromatography was used to separate the steroid products, and gas chromatography-mass spectrometry was applied for the chemical identification of the product. 7alpha-Hydroxypregnenolone, previously known to be produced only by ovarian tissues, was found to be the sole metabolite of pregnenolone metabolism by rainbow trout embryos. Sulfate and glucuronide conjugated forms of 7alpha-hydroxypregnenolone were also produced. We hypothesize that this metabolite provides a pathway for excretion of pregnenolone, enabling the embryo to maintain its own steroid milieu, although the possibility of 7alpha-hydroxypregnenolone also playing a physiological role cannot be excluded.

Evidence of a high percentage of intersex in the Mediterranean swordfish (Xiphias gladius L.).

The first evidence of the presence of intersexuality in a wild population of Mediterranean swordfish (Xiphias gladius L.) is reported. Forty of 162 specimens (25%) macroscopically classified as males, showed the presence of female germ cells within the testes. In two specimens grouped previtellogenic oocytes were present; all the other specimens possessed single scattered previtellogenic oocytes. The presence of vitellogenin was demonstrated immunohistochemically in the liver of both intersex and normal males. These findings could be due to the exposure to oestrogen-mimicking substances.

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.

Wild intersex roach (Rutilus rutilus) have reduced fertility.

Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = -0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.

The use of motility as a measure of sperm quality in fish is reviewed. Computer assisted sperm analysis (CASA) provides a simple and rapid quantitative assessment of the quality of fish sperm and may predict its ability to fertilize eggs. It has been used to: monitor the effects of heavy metal pollutants, such as mercury and tributyltin, on sperm quality; to select broodstock; to improve the efficiency of cryopreservation and storage; and to optimise conditions for fertilisation. In combination with CASA, morphological measurements can be used to determine the causes of reduced sperm motility. Technical details for the use of CASA are described.

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus).

A new integrated approach including computer-assisted sperm analysis (CASA), viability staining and fertilization was used to study the quality of cryodiluents used in fish sperm cryopreservation. As an example the sperm quality of an African catfish, Clarias gariepinus (Burchell, 1822), was assessed by its fertilizing ability, motility and viability at day 0 (fresh), after 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months frozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solutions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3, M4) associating 10% hen's egg yolk. Progressive sperm movement measured by CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated with hatching rates obtained from fertilization using minimal sperm:egg ratios. After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yolk had deteriorated less than that of spermatozoa stored at 4 degreesC. Post-thaw hatching rates reflected the post-thaw sperm viability, which was cryodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) and 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motility of 10-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64.0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after activation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) between 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality measurements and at all freezing durations. Sperm motility was positively related to fertility. Our method will make it possible to develop even better extenders and cryoprotectants.

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.

Synthesis of 17,20alpha/beta-dihydroxy-4-pregnen-3-one and 5beta-pregnanes in spermatozoa of primary and 17alpha-methyltestosterone-induced secondary male grouper (Epinephelus coioides).

The in vitro metabolism of [3H]17-hydroxyprogesterone by spermatozoa of primary and 17alpha-methyltestosterone (MT)-induced secondary male grouper (Epinephelus coioides) has been examined. With only the labeled precursor, the four predominant 5beta-pregnanes (17, 20alpha-dihydroxy-5beta-pregnan-3-one; 5beta-pregnan-3alpha,17, 20alpha-triol; 3alpha,17-dihydroxy-5beta-pregnan-20-one; and 17hydroxy-5beta-pregnan-3,20-dione) accounted for 75% of the total product yield, with the remainder comprising 17, 20alpha-dihydroxy-4-pregnen-3-one (17,20alphaP) and traces (3%) of 17,20beta-dihydroxy-4-pregnen-3-one. This is the first report of the synthesis of these 5beta-pregnanes by teleost spermatozoa. The addition of excess unlabeled precursor caused a marked shift to the synthesis of only 17,20alphaP. The steroidogenic profile in spermatozoa of the primary and secondary males appears similar in both cases. The shift to 17,20alphaP synthesis at high substrate concentration may suggest a gonadotropin (GtH) control of 17, 20alphaP synthesis, involving the competition for 17P between the high-activity, low-capacity 5beta-reductases and the low-activity, high-capacity 20alpha-HSD. Poor spermiation in MT-induced males may thus be due to the lack of 17P arising from low GtH secretion in vivo in the secondary males.

Extragonadal production of 17,20-dihydroxy-4-pregnen-3-ones in vitro in cyprinid fish.

Muscle, heart, eye, testis, blood, and sperm of trout and goldfish have been examined for the presence of 20-hydroxysteroid dehydrogenase by incubation with [3H]17-hydroxyprogesterone in the presence of 0, 0.1, 1, or 10 micrograms/ml of unlabeled substrate. In trout, 17-hydroxyprogesterone was metabolized only by sperm and testis, with 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) as the major product and traces (< 2%) of 17,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha P). In goldfish, maximum conversion of 17-hydroxyprogesterone to 17,20 alpha P was over 30% per 100 mg tissue in eyeball, heart, and testis, 12% per 20 microliters blood, and 18% per 20 microliters sperm, but less than 5% per 100 mg muscle. 17,20 alpha P was the only metabolite of 17-hydroxyprogesterone in all incubations of nongonadal tissue in goldfish. Within the cyprinid eye, there was no significant activity in the lens, the fluids, or the retina. The possible relationship between the teleost 20 alpha- and 20 beta-hydroxysteroid dehydrogenases, the mammalian 20-hydroxysteroid dehydrogenases, and members of the aldo-keto reductase and short-chain dehydrogenase superfamilies is discussed.

In vitro effects of gamma-hexachlorocyclohexane on in vitro biosynthesis and metabolism of steroids in goldfish Carassius auratus.

Testicular and ovarian fragments of Carassius auratus, taken during the reproductively active prespawning phase (June) of its annual reproductive cycle, were incubated with different concentrations (0 mg/ml, 0 ppm; 0.001 mg/ml, 1 ppm; 0.01 mg/ml, 10 ppm; and 0.02 mg/ml, 20 ppm) of gamma-hexachlorocyclohexane (gamma-HCH) in the presence of either exogenous precursor [3H]-17-hydroxyprogesterone ([3H]17-P) or carp hypophyseal homogenate. The free (unconjugated) and conjugated metabolites (glucuronides and sulfates) of [3H]-17-P [androstenedione (AD), androstenetrione, 17-hydroxyprogesterone, testosterone (T), 11-deoxycortisol (S), 17, 20alpha-dihydroxy-4-pregnen-3-one (17,20alphaP), 17, 20beta-dihydroxy-4-pregnen-3-one (17,20betaP), 7alpha-pregnanetetrols (7alpha-P), and other polar metabolites] were separated by thin-layer chromatography and high-performance liquid chromatography. The endogenous production of unconjugated (free) steroids T, 17,20betaP, S, and 11-ketotestosterone (11-KT) in response to gamma-HCH were measured by radioimmunoassay. Among the in vitro metabolism of [3H]-17-P, in males, free steroids of AD, T, 17,20alphaP, S, and polar-free steroids were increased with the decreased yield of 11-KT. Percentage yield of testosterone glucuronide (TG) was increased with highly significant decreased yields of polar glucuronide steroids. The sulfate steroids of 17, 20alphaP, 17,20betaP, S, and 11-KT remain unchanged. In females, the decreased percentage of yield of AD and S and elevated T were noticed. The yield of TG was increased with decreased yield of 7alpha-P glucuronides. The percentage yield of AD sulfate and sulfate steroids of 17,20alphaP, 17,20betaP, and S were noted to be increased, but the yield for S sulfate was very high. Endogenous production of T was increased in both sexes in the presence of gamma-HCH, but 11-KT in male and S in female were depressed. 17, 20betaP was stimulated at some concentrations in both sexes but levels were very low. Results indicate that gamma-HCH in vitro perturbed the steroid biosynthesis during this phase thereby affecting reproductive physiology.

In vitro biosynthesis of androgens in the Australian lungfish, Neoceratodus forsteri.

The serum concentration of testosterone was estimated from a population of wild lungfish over 6-7 years of sampling. Male lungfish were found to have high circulating levels of testosterone (approximately 50 ng/ml) which varied seasonally and could be correlated with spermatogenesis as judged by testis histology. Incubation of testis tissue slices with [3H]progesterone, [3H]17-hydroxyprogesterone, or [3H]testosterone confirmed that testosterone is the major androgen in Neoceratodus. Not even trace amounts of 11-keto- or 11 beta-hydroxytestosterone or 5 alpha-dihydrotestosterone could be identified by TLC separations. There was little or no conjugation of steroids by the testes, except during the spawning season, when glucuronides of androstenedione and testosterone were produced.

Biosynthesis of 17,20 alpha-dihydroxy-4-pregnen-3-one, 17,20 beta-dihydroxy-4-pregnen-3-one, and 11-ketotestosterone by testicular fragments and sperm of the roach, Rutilus rutilus.

The effect of incubation time and substrate concentration on the in vitro metabolism of 17-hydroxyprogesterone by testes of the roach has been examined. There was a shift from synthesis of the 11-oxygenated androgens, 11-ketotestosterone, and androstenetrione at low substrate concentration to 17,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha P) at high substrate. Glucuronides and sulfates were of significant importance only at low substrate and long incubation times. There was a shift from 17,20 beta-dihydroxy-4-pregnen-3-one to 17,20 alpha P with increased substrate. The results confirm that substrate affects the steroidogenic profile and that 3 hr is optimal time for such studies. Incubations of sperm with 17-hydroxyprogesterone gave predominantly 11-ketotestosterone at low substrate concentrations and 17,20 alpha P at high substrate. The synthesis of 11-ketotestosterone is demonstrated for the first time in teleost sperm.

In vitro metabolism of testosterone by gonads of the grouper (Epinephelus tauvina) before and after sex inversion with 17 alpha-methyltestosterone.

The steroidogenic potential of gonadal tissues of the protogynous grouper (Epinephelus tauvina) was examined before and after sex inversion with 17 alpha-methyltestosterone. Incubations of gonadal tissues of the fish with [3H]testosterone resulted in the biosynthesis of 5 beta-androstane-3 beta, 17 beta-diol and 5 beta-dihydrotestosterone as the only metabolites in the female phase. Although the production of these 5 beta-reduced androgens persisted in the male phase, there was a shift toward the production of 11 beta-hydroxytestosterone and 11-ketotestosterone as major metabolites.

Increased substrate concentration causes a shift from production of 11-oxygenated androgens to 17,20-dihydroxyprogestogens during the in vitro metabolism of 17-hydroxyprogesterone by goldfish testes.

Goldfish testes were incubated with [3H]17-hydroxyprogesterone in the presence of 0 to 100 micrograms/ml of unlabeled substrate and metabolites examined by thin-layer and high performance liquid chromatography. Conjugated steroids, predominantly sulfates, accounted for 50% of recovered activity with radiolabeled substrate alone, but percentage yields decreased to very low levels with substrate concentrations of 1 micrograms/ml and above. The 11-oxygenated androgens, androstenetrione and 11-ketotestosterone, were the major products with 0 to 0.1 micrograms/ml substrate, but at concentrations of 1 to 100 micrograms/ml the major products were 17,20 alpha-dihydroxy-4-pregnen-3-one (30% of recovered activity) with smaller amounts of the 20 beta-epimer. 11-Deoxycortisol was a minor product at all substrate concentrations. Production of 11-oxygenated androgens in the medium reached a maximum value of 40 ng/100 mg tissue/3 hr with 2 micrograms substrate, but progestogen production continued to increase up to the maximum substrate used (30 micrograms at 200 micrograms substrate). The results demonstrate a clear switch from production of 11-oxygenated androgens to that of 20-reduced progestogens with increased substrate concentration. This switch shows similarities to that observed for in vivo plasma steroid concentrations during the prespawning period of many male teleosts and it is suggested that this, at least in part, may be due to increased substrate availability resulting from elevated gonadotropin secretion.

The in vitro metabolism of 17-hydroxyprogesterone by ovaries of the goldfish, Carassius auratus, is affected by substrate concentration.

Ovaries of mature goldfish (Carassius auratus) were incubated with 17-[3H]hydroxyprogesterone and 0, 0.01, 0.1, 1, 10, and 100 micrograms/ml 17-hydroxyprogesterone (17P). The major products were quantified after separation by thin-layer and high-performance chromatography. The results show that substrate concentration markedly affects the pattern of metabolites. Glucuronides and sulfates decreased from 7.2 +/- 2.0 and 28.5 +/- 4.4% of recovered radioactivity, respectively, in the absence of added 17P to insignificant yields at 10 micrograms/ml 17P. 7 alpha-Tetrols were the major products (36-52%) with 0-1 microgram/ml substrate, but at 10 and 100 micrograms/ml 17,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha P) was the major metabolite and there were low yields of other products. 11-Deoxycortisol was produced in significant amounts only at low substrate concentrations. The results support the hypothesis that 5 alpha-reduced-7 alpha-hydroxylated metabolites are formed by enzymes of high activity but low capacity and serve to limit secretion of 17,20 alpha P and its 20 beta epimer to the period of maximum GtH stimulation.

Substrate concentration affects the in vitro metabolism of 17-hydroxyprogesterone by ovaries of the carp, Cyprinus carpio.

Carp ovarian tissue was incubated with (3)H-17-hydroxyprogesterone in the presence of 0, 0.1, 1, 10, and 100 µg ml(-1) unlabeled 17-hydroxyprogesterone. The pattern of metabolites formed showed a marked variation with substrate concentration. Formation of glucuronide and sulphate conjugates was important only at low substrate concentration. At high substrate concentration (10 and 100 µg ml(-1)) 17,20α-dihydroxy-4-pregnen-3-one was the major metabolite, but at intermediate concentrations polar 7α-hydroxypregnanetetrols predominated. The results support the hypothesis that at low substrate concentrations conjugating, 5α-reducing and 7α-hydroxylating enzymes, of high activity but low capacity, act as scavengers to deactivate any steroids formed during the relatively low pituitary gonadotrophin secretions which are necessary for oocyte development, but that during the prespawning gonadotrophin surge when high levels of substrate are present these enzymes are saturated and 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) becomes the major ovarian steroid. The possible role of 17,20αP during oocyte final maturation requires further examination.

Modulatory actions of Mystus gonadotropin on gamma-BHC-induced histological changes, cholesterol, and sex steroid levels in Heteropneustes fossilis.

Female specimens of Heteropneustes fossilis were exposed to a sublethal concentration of the organochlorine agricultural pesticide gamma-BHC (16 mg liter-1) for 4 weeks during the reproductively active preparatory and prespawning phases of the annual reproductive cycle. The effects of gamma-BHC on the gonadosomatic index (GSI) and on the histological changes in ovary, cholesterol-free cholesterol (CF), esterified cholesterol (CE), and the sex steroids testosterone-estradiol-17 beta, and 17 alpha-hydroxyprogesterone in plasma and ovary were monitored. Mystus gonadotropin (mGTH) was administered along with gamma-BHC exposure during the preparatory and prespawning phases. gamma-BHC decreases GSI during both phases; however, simultaneous administration of mGTH recovered the effect. gamma-BHC exposure caused inhibition of ovarian growth, resulting in a large number of stage I oocytes and few stage II oocytes, while at higher doses, mGTH reversed the impact of gamma-BHC, and a large number of stage III and vitellogenic oocytes were recorded. This pesticide also inhibited deesterification of CE to CF. Additionally, it impaired ovarian steroidogenesis and secretion of steroids into plasma. Lower doses of mGTH (0.1 and 1.0 microgram/fish) were ineffective in modulating the impact of gamma-BHC on lipids and steroids. However, higher doses (10 and 20 micrograms/fish) effectively revoked the impact of gamma-BHC, routed through the hypothalamo-hypophyseal-ovarian axis and directly upon the ovary.