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.

Steroid metabolism in teleost gonads: purification and identification of metabolites by high-performance liquid chromatography.

A simple, efficient, and comprehensive technique for the purification, identification, and quantitation of the common steroid metabolites synthesized by the gonads of teleosts involving five systems of high-performance liquid chromatography (HPLC) was developed. Steroid standards were identified in HPLC by UV absorption at 254 nm or 280 nm, by differential refractive index, or by using radioactive standards. Metabolites that do not absorb UV light and are not resolved in the isocratic HPLC systems were identified in thin-layer chromatography following purification by HPLC. By using this technique, most of the steroid metabolites, including some polar metabolites, synthesized by the gonadal tissues of the teleosts can be purified within three steps of chromatography. The HPLC systems reported here are also useful in identifying the chromium trioxide oxidized products of metabolites, such as triols and tetrols, which considerably narrows down the number of probable metabolites.

The effect of cadmium on steroidogenesis by testes of the rainbow trout, Salmo gairdneri.

Testes of the rainbow trout were incubated in the presence of 0, 0.05, 0.5 and 5 mM cadmium (Cd). Production of those steroids measured was stimulated by 0.05 and 0.5 mM cadmium. Yields of testosterone (T) and 11 beta-hydroxytestosterone (beta) were significantly increased at these concentrations, but there was little effect on glucuronyl transferase and 11 beta-hydroxysteroid dehydrogenase enzyme activities. Cd-induced alteration of the hormonal balance may result in perturbation of reproductive development.

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.

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.

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.

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.

Progestogen metabolism by ovaries of the roach (Rutilus rutilus L.) and the rudd (Scardinius erythrophthalmus L.).

Roach ovaries converted 17-hydroxyprogesterone to 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) and to glucuronides of testosterone and 17,20αP. Small amounts of 5α-pregnane-3α- and -3ß, 17, 20α-triols, 7α-hydroxy-5α-reduced metabolites and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) were also formed. Rudd ovaries converted this substrate mainly to 17,20αP, 5α-pregnane-3α- and -3ß,17,20α-triols, 17,20α-dihydroxy-5α-pregnan-3-one and testosterone glucuronide. The main metabolites of progesterone with both species were 17,20αP, 5α-pregnane-3ß,17,20α-triol and 7α-hydroxy-5α-reduced steroids. Rudd ovaries formed, in addition, 17,20α-dihydroxy-5α-pregnan-3-one from progesterone. The pattern of metabolites was markedly altered when the concentration of substrate was increased from 42ng to 1 µg or 100 µg. At the highest concentration, glucuronides and polar steroids were not detectable, while at low concentrations they accounted for over 50% of the metabolites. 20α-Hydroxysteroid dehydrogenase was shown to have a very high capacity, producing 21-47 µg 17,20αP from 100 µg 17-hydroxyprogesterone substrate with 200 mg ovarian tissue in 5h.

In vitro metabolism of progesterone, 17-hydroxyprogesterone, and 17,20 beta-dihydroxy-4-pregnen-3-one by ovaries of the common carp Cyprinus carpio: production rates of polar metabolites.

[14C]Progesterone, 17-[3H]hydroxyprogesterone, and 17,20 beta-[3H]dihydroxy-4-pregnen-3-one (17,20 beta P) were incubated for 1, 3, 6 or 24 hr with ovaries from carp which had received injections of either carp pituitary extract or saline. All three substrates were very rapidly metabolized to polar products, but 17,20 beta P was not detected as a metabolite of either progesterone or 17-hydroxyprogesterone. The major metabolite in all incubations was very similar, but not identical, in chemical and chromatographic properties to 5 beta-pregnane-3 alpha, 6 alpha, 17,20 beta-tetrol. A compound isopolar with 5 beta-pregnane-3 alpha, 6 alpha, 17,20 alpha-tetrol was isolated only in incubations of progesterone and 17-hydroxyprogesterone. It is suggested that hydroxylation and reduction may be a deactivation mechanism allowing 17,20 beta P to build up as an intermediate, and active maturation inducing steroid, only in response to the ovulatory gonadotrophin surge, thus assisting in synchrony of oocyte maturation.

Gonadotropin-induced changes in steroid production by ovaries of the common careCyprinus carnio L. around the time of ovulation.

Ovarian fragments from both primed (gonadotrophin treated) and unprimed female carp were incubated either with or without carp hypophysial homogenate and steroid hormone production measured. In incubations without hypophysial homogenate, production of all the steroids measured was either very low or nondetectable and there was no significant difference between tissue from primed and unprimed fish. In the presence of carp hypophysial homogenate a very significant increase in production of testosterone, 17-hydroxyprogesterone and testosterone glucuronide was observed, but there was no significant difference between primed and unprimed fish. 17,20ß-Dihydroxy-4-pregnen-3-one (17,20ßP) was not stimulated by carp hypophysial homogenatein vitro in ovaries from unprimed fish, but a very significant increase in production of this hormone was observed in tissue from fish which had received a priming dose of pituitary hormone. It is suggested that the priming dose of pituitary extract used in the normal hypophysation procedure to induce ovulation in teleosts initiates the potential for synthesis of 17,20ßP in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.

Temperature regulation of ovarian steroid production in the common carp, Cyprinus carpio L., in vivo and in vitro.

The effect of temperature on ovarian steroid production in the common carp, Cyprinus carpio L., has been studied in vitro with exogenous and endogenous precursors, and in fish held at three different temperatures in vivo. With radioactive testosterone as substrate, the major metabolite was testosterone glucuronide, but androstenedione and 5 alpha-androstane-3 beta,17 beta-diol were also identified. 5 alpha-Androstane-3 beta,7 alpha,17 beta-triol was tentatively identified and two other polar metabolites were isolated, one of which was convertible to this triol. A significant increase in production of most metabolites occurred between 20 and 24 degrees. Production of estradiol and testosterone from endogenous substrate under gonadotrophin stimulation in vitro showed a marked temperature dependence, but the response was closely related to ovarian maturity. Stage 4-5 ovaries produced testosterone, while late Stage 3 tissue produced only estradiol. Neither steroid was produced in significant quantities by less mature ovaries. The results indicate that the "switch off" of ovarian aromatase activity at the end of vitellogenesis is actuated by an ovarian rather than by a pituitary factor. Secretion of testosterone and estradiol showed a very significant change with temperature with the optimum at 24-29 degrees. Profiles for individual fish show that this optimal range is extremely narrow, particularly for estradiol, where secretion may increase as much as twentyfold over 5 degrees. The results in general correlate well with 24 degrees as the most favourable temperature for reproduction in carp. Plasma concentrations of testosterone and estradiol closely paralleled the in vitro secretion rates of these hormones. Plasma testosterone levels were greatest in the most mature fish, whereas plasma estradiol was significantly higher in late Stage 3 fish than in those of greater or lesser ovarian maturity. More Stage 4 and 5 fish were found in the group held at 24 degrees than at 20 or 29 degrees for 4 weeks, but all groups contained a high proportion of early Stage 3 fish.

The effect of temperature on steroid biosynthesis by testes of the dogfish, Scyliorhinus caniculus.

1. Testes of the dogfish, Scyliorhinus caniculus, were incubated with [3H]pregnenolone and [3H]testosterone at a range of temperatures from 1 to 36 degrees C. 2. Yields of testosterone from pregnenolone were maximal between 11 and 16 degrees C, corresponding to the breeding temperature of the species. 3. Yields of glucuronides and sulphates increased from 1 to 26 degrees C. 4. Yields of an unidentified polar metabolite increased with temperature and it is suggested that this may play a regulatory role in elasmobranch reproduction.

Autoradiographic localization of gonadotrophin receptors in ovaries of the common carp, Cyprinus carpio L.

Binding sites for carp gonadotrophin have been located in carp ovaries using [125I]labeled gonadotrophin and autoradiography. The radioactive gonadotrophin was displaced from tissue by unlabeled gonadotrophin or carp hypophysial homogenate in a dose-dependent fashion. No binding of gonadotrophin was found in previtellogenic oocytes but binding appeared with the first indications of vitellogenesis. In the smaller vitellogenic oocytes binding was uniformly distributed in the follicular envelope, but in the largest oocytes binding was restricted to the interstitial tissue. In these more mature oocytes gonadotrophin was also found within the oocyte and appeared to migrate toward the nucleus. The relationship between binding location, steroidogenesis, and oocyte maturation is discussed. We found no evidence for specific binding of [125I]thyroxine under comparable conditions.

Effect of gonadectomy and hypophysectomy on plasma steroid levels in male and female lampreys (Lampetra fluviatilis, L.).

Testosterone, estradiol, and dehydroepiandrosterone (T, E2, and DHA) were measured by RIA in plasma samples from three groups of sexually immature male and female (male, female) river lampreys: (I) intact, (II) gonadectomized, or (III) with pro- and mesoadenohypophysis removed (hypophysectomized). T levels were around 0.1 ng/ml in both I male and I female and increased significantly in all operated groups, especially in II male, to 1.2 ng/ml. E2 levels were between 0.7 and 1 ng/ml in I male and I female and were significantly elevated only in II male to 2.0 ng/ml. DHA levels showed significant decreases, from around 0.9 ng/ml in I male and I female, in both operated groups. It is suggested that T and E2 are secreted from a nongonadal source in both sexes and then converted to biologically active sex hormones in the gonads. Therefore T and E2 do not decrease, and may even accumulate, in the blood after gonadectomy. Since extirpation of the pro- and mesoadenohypophysis was not followed by a decrease in T and E2, their secretion may be stimulated by a hormone from another part of the pituitary such as the metaadenohypophysis.

The effect of temperature and gonadotropin on testicular steroidogenesis in Sarotherodon (Tilapia) mossambicus in vitro.

Testes of sexually mature Sarotherodon mossambicus were incubated at 15, 22, 30, and 40 degrees with (a) tritiated testosterone and (b) salmon pituitary extract. Formation of 11-keto- and 11 beta-hydroxytestosterone from the tritiated precursor showed little change in yield between 15 and 30 degrees but yields of glucuronides rose dramatically between 22 and 30 degrees and a significant rise was observed for formation of 5 beta-androstane-3 alpha, 17 beta-diol between 15 and 40 degrees. Yields of 3 alpha, 17 beta-dihydroxy-5 beta-androstan-11-one followed a pattern similar to that of 11-ketotestosterone. With endogenous precursors under the stimulation of salmon pituitary extract, yields of testosterone, 11-ketotestosterone, and 11 beta-hydroxytestosterone were maximal at 22 degrees after which they declined to very low levels at 40 degrees. Yields of testosterone and 11-ketotestosterone glucuronides while showing a peak at 22 degrees declined much more slowly at higher temperatures than did those of the free steroids. In the absence of pituitary stimulation, levels of all steroids were below the limits of detection. Plasma levels of testosterone (15.3 +/- 1.5 ng/ml), 11-ketotestosterone (5.3 +/- 2.7 ng/ml), 11 beta-hydroxytestosterone (5.5 +/- 2.6 ng/ml), and their glucuromides (1.5 +/- 0.5, 0.14 +/- 0.1, and 1.5 +/- 0.5 ng/ml, respectively) were measured in fish held at 25 degrees. A rapid conchromatographic method for the assay of the three free steroids is described and the results are shown to be comparable to those obtained after chromatography.

Hormonal changes during induced ovulation of the carp, Cyprinus carpio.

The changes in plasma concentrations of eight steroids (testosterone, testosterone glucuronide, estradiol, estradiol glucuronide, 17-hydroxyprogesterone, 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P), deoxycorticosterone, and cortisol) have been followed in three individual carp, Cyprinus carpio, during ovulation induced by carp pituitary extract. Deoxycorticosterone and estradiol glucuronide were not detectable and small amounts of 17,20 beta P were found only in one fish. A priming injection of pituitary extract, administered 24 hr before ovulation, stimulated an increase in testosterone 3-6 hr later, followed by an increase in estradiol. The second injection of pituitary extract given 12 hr after the priming dose, resulted in a second peak of testosterone which was accompanied by a peak of testosterone glucuronide, a conjugate which was usually formed in only small amounts following the priming dose. A sharp peak of 17-hydroxyprogesterone followed the second stimulation and it is suggested that this, rather than 17,20 beta P, may be the natural inducer of maturation in carp. Although cortisol levels showed large variations, they appeared to be stimulated by the priming dose of pituitary extract. Levels of all steroids, except for cortisol, fell rapidly after ovulation.

The effect of temperature on testicular steroid production in the rainbow trout, Salmo gairdneri, in vivo and in vitro.

The effect of temperature on steroidogenesis in the male rainbow trout has been studied both in vitro using endogenous precursors under gonadotrophin stimulation and in vivo in fish held for 2 weeks at three different temperatures. In vitro, the optimum temperature for formation of testosterone and its 11-oxygenated derivatives was 10 degrees, whereas glucuronide formation showed an optimum at 18 degrees. In vivo, plasma levels of testosterone and 11-keto-testosterone were significantly higher at 6 than at 17 degrees, whereas glucuronide levels showed no significant difference. Milt was obtained only from fish held at 6 and 12 degrees. The optimum temperature for free steroid formation in response to gonadotrophin stimulus is shown to be dependent upon glucuronyl transferase content, and its progressive increase during the reproductive cycle may provide a mechanism for the inhibition of free steroid synthesis and hence spermiation at elevated temperatures where gamete survival is poor.

Steroidogenesis by gonads of a viviparous teleost, the sailfin molly (Poecilia latipinna), in vitro and in vivo.

Gonads of Poecilia latipinna transformed [3H]testosterone into a number of reduced and conjugated metabolites in high yield in vitro. In the male 5 beta-androstane-3 alpha, 17 beta-diol, 5 beta-androstane-3 beta, 17 beta-diol, and their "sulphates" were identified. The only 11-oxygenated androgen detected was a compound tentatively identified as 5 beta-androstane-3 beta, 11 beta, 17 beta-triol. In ovarian incubates androstenedione, 5 beta-androstane-3 alpha, 17 beta-diol and its glucuronide, testosterone glucuronide, and 5 beta-androstane-3 alpha, 17 beta-diol glucuronide were identified. Highest yields of the ovarian glucuronides coincided with the termination of vitellogenesis which may indicate a possible pheromonal role of these conjugates. In vivo plasma levels of estradiol in the female were correlated with vitellogenesis and fell markedly after castration or hypophysectomy. In males the plasma concentrations of testosterone, 11-ketotestosterone and 11 beta-hydroxytestosterone and their conjugates were variable and not apparently correlated with testicular weight, but they were reduced to undetectable levels by castration and hypophysectomy. The results suggest that 5 alpha- and 5 beta-reduced steroids may play a role in the reproductive endocrinology of P. latipinna and that measurements of only the "classical" steroid hormones in this and possibly other species may give only a partial and misleading picture of endocrine changes.

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 synthesis of 20α-reduced and of 11- and 21-oxygenated steroids and their sulfates by testes of the goldfish (Carassius auratus): Testicular synthesis of corticosteroids.

Testes from spermiating goldfish were incubated with [(3)H]17-hydroxyprogesterone. The major products in the unconjugated fraction were identified as androstenedione, androstenetrione, 11-ß-hydroxyandrostenedione, 11-ketotestosterone, 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) and 11-deoxycortisol. Testosterone was present predominantly in the glucuronide fraction, but yields were low (1-3%). The major components of the sulfate fraction were 17,20αP and 11-deoxycortisol. The identification of cortisone in low yield (< 2.5010) in both the free and sulfate fractions is the first report of corticosteroid biosynthesis by a teleost testis. The high yields of 17,20αP and 11-deoxycortisol and their sulphates suggests that their possible role in spermiation of the goldfish should be further investigated.