Plasma Sex Steroid Research Articles

Plasma Sex Steroid Levels and Steroidogenesis in the Gonad of the Self-fertilizing Fish Rivulus marmoratus

The mangrove killifish, Rivulus marmoratus, is the only known self-fertilizing vertebrate. This species is sexually dimorphic; sexually mature individuals are either hermaphrodite or primary and secondary males. Although the mangrove killifish has a unique reproductive strategy, there has been no study on the reproductive endocrinology of this species. Thus we investigated plasma sex steroid hormone levels and steroidogenesis in the gonads of R. marmoratus by enzyme linked immunosorbent assay (ELISA). Plasma 17β-estradiol (E2) and 11-ketotestosterone (11-KT) were detected both in hermaphrodite and in primary male. Ovarian follicles (follicle-enclosed oocytes) from hermaphrodites, which were categorized into early yolk stage and late yolk stage, and testis tissue of primary males were cultured with different concentrations of 17α-hydroxyprogesterone (OHP) or testosterone (T) for 24 h. Production of T, E2, 11-KT and 17α-20 β-dihydroxy-4-pregnen-3-one (17α,20β-P) in the medium from tissue culture were measured by ELISA. Early and late ovarian follicles of hermaphrodites and testis pieces of primary males synchronously secreted E2, 11-KT, and 17α,20β-P following incubation with OHP or T. We conclude that both hermaphrodite and primary male of the mangrove killifish secrete estrogen, androgen, and progestin synchronously.

Breast cancer, menopause, and long-term survivorship: critical issues for the 21st century

Breast cancer accounts for 33% of all incident cancers in women in North America, and there are an estimated >2 million breast cancer survivors in the United States today. Ovarian hormones are intimately involved in the initiation and promotion of breast cancer development, with targeted endocrine therapies being the most widely used as anticancer treatment. It is not surprising that these treatments frequently cause persistent menopausal symptoms in breast cancer survivors. In addition, adjuvant chemotherapy often induces premature menopause in younger patients with breast cancer. Some women at high risk for the development of breast cancer (e.g., precancerous breast disease, carriers of deleterious hereditary predisposition genes) experience vasomotor symptoms as a result of tamoxifen therapy or preventive oophorectomy. Clinical management of menopausal symptoms in these settings is complicated by the relative prohibition of hormonal therapies and the fact that breast cancer-directed therapies often exacerbate these menopausal symptoms. Thus, this special population of women requires unique management strategies and deserves separate consideration.

Seasonal Variations in Plasma Concentrations of Sex Steroid Hormones and Vitellogenin in Wild Male Japanese Dace (Tribolodon hakonensis) Collected from Different Sites of the Jinzu River Basin

In order to clarify the seasonal variations of plasma sex steroid hormones and vitellogenin (VTG) concentrations in the wild male Japanese dace, Tribolodon hakonensis, we measured plasma levels of testosterone (T), 11-ketotestosterone (11-KT), estradiol-17 beta (E2) and VTG, as well as spermatogenetic stages and gonadosomatic index (GSI). Wild Japanese dace were collected from different sites of the Jinzu River basin (including the Takahara River and the Itachi River). The fish from Toyama Bay were also measured the spermatogenetic stages, GSI and VTG levels. The seasonal variations of the hormone levels were discussed in the relationship with various environmental factors. In landlocked fish of the Takahara River, the plasma concentrations of T and E2 reached the highest levels in May and June. In the fish collected from the Itachi River, plasma concentrations of T, 11-KT and E2 reached the highest levels during breeding season of April and May. Sexual maturation, evaluating from GSI and the spermatogenetic stages, proceeded earlier in the fish population at Toyama Bay, and afterward it was followed in the fish population at the Takahara River, in associated with a rise of environmental water temperature at fish captured sites. In the male dace, low but detectable levels of plasma E2 were measured and there were significantly positive correlations between E2 level and the levels of GSI, VTG or T. These results suggest that E2 might be a necessary sex steroid hormone related to gonad maturation, and that circulating E2 may induce VTG production in the wild male Japanese dace.

Sympatric rattlesnakes with contrasting mating systems show differences in seasonal patterns of plasma sex steroids

Long-term field studies conducted in Arizona show that two species of sympatric rattlesnakes differ in the structure of their mating systems, primarily in frequency and timing of mating seasons, despite exposure to identical environmental conditions. The western diamond-backed rattlesnake, Crotalus atrox, has two distinct mating seasons within a single annual spermatogenic cycle. The first mating season occurs from late August to early October. Following a hibernation period of 4 months, the second mating season occurs from mid-March to early May. Because there is a mating season in spring, long-term sperm storage by females during winter is facultative. In contrast, the black-tailed rattlesnake, Crotalus molossus, has a single mating season (mid-July to early September) within a single annual spermatogenic cycle. Due to the absence of a mating season in spring, long-term sperm storage by females during winter is obligatory. In both species, ovulation and fertilization occur in spring, and offspring are produced from mid-July to early September. Based on these robust data, we tested the hypotheses that seasonal patterns of plasma sex steroids (testosterone, 5α-dihydrotestosterone, and 17β-estradiol) differ between males in wild populations of C. atrox and C. molossus, and that peak levels would be coincident with the mating seasons. Specifically, we predicted that there would be two peaks of sex steroids in C. atrox and one peak in C. molossus, and that baseline levels would be detected outside the periods of mating and spermatogenesis. Our results supported these predictions. Furthermore, absolute concentrations of plasma testosterone and 5α-dihydrotestosterone, but not 17β-estradiol, were higher in C. atrox than in C. molossus. We discuss a possible scenario for the evolution of the different mating seasons in these sympatric rattlesnakes, and advocate that comparative approaches to address such questions should integrate proximate and ultimate causation to increase explanatory power.

Effects of Atrazine on CYP19 Gene Expression and Aromatase Activity in Testes and on Plasma Sex Steroid Concentrations of Male African Clawed Frogs (Xenopus laevis)

Some investigators have suggested that the triazine herbicide atrazine can cause demasculinization of male amphibians via upregulation of the enzyme aromatase. Male adult African clawed frogs (Xenopus laevis) were exposed to three nominal concentrations of atrazine (1, 25, or 250 microg atrazine/l) for 36 days, and testicular aromatase activity and CYP19 gene expression, as well as concentrations of the plasma sex steroids testosterone (T) and 17beta-estradiol (E2), and gonad size (GSI) were measured. There were no effects on any of the parameters measured, with the exception of plasma T concentrations. Plasma T concentrations in X. laevis exposed to the greatest concentration of atrazine were significantly less (p = 0.034) than those in untreated frogs. Both CYP19 gene expression and aromatase activities were low regardless of treatment, and neither parameter correlated with the other. We conclude that aromatase enzyme activity and gene expression were at basal levels in X. laevis from all treatments, and that the tested concentrations of atrazine did not interfere with steroidogenesis through an aromatase-mediated mechanism of action.

Seasonal variation in plasma thyroxine concentrations in juvenile alligators ( Alligators mississippiensis) from three Florida Lakes

Circulating concentrations of thyroxine (T 4) vary seasonally in many vertebrates. This study examined the seasonal variation in plasma concentrations of T 4 in juvenile American alligators ( Alligator mississippiensis) from three populations in central Florida, USA. One site, Lake Woodruff National Wildlife Refuge, is considered a reference site whereas the other two lakes, Lake Apopka and Orange Lake, are significantly impacted by human activity. Juvenile American alligators ranging from 75–150 cm in total length were hand-captured at night from November 2000–April 2002. Plasma thyroxine concentrations were analyzed using a radioimmunoassay (RIA) previously validated for alligator plasma. Juvenile American alligators display seasonal variation in circulating T 4 concentrations. Plasma T 4 concentrations decrease from August/September to November and then begin a slow rise until April, at which point they plateau. Sex of juveniles influenced plasma concentrations of T 4 in some months but did not appear to alter the pattern in seasonal variation. The pattern we observed in plasma T 4 concentrations is not directly related to an environmental factor such as ambient temperature but is similar to that seen in plasma sex steroid concentrations during the reproductive cycle of adult alligators. Although the pattern and plasma concentration of T 4 exhibits significant variation among the three lakes studied, the pattern in seasonal variation appears similar. Comparing the seasonal pattern in plasma T 4 with plasma concentrations of sex steroids (testosterone and estradiol-17β) or corticosterone could provide important information on the peripubescent life stage of the American alligator.

Plasma sex steroid and thyroid hormones profile in male water frogs of the Rana esculenta complex from agricultural and pristine areas

Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa ( Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17β, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17β peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.

Parasitism in fish—an endocrine modulator of ecological relevance?

Wild bream ( Abramis brama) were collected from the river Elbe, and the influences of parasitic infection by the tapeworm Ligula intestinalis on endocrine and related functions (vitellogenin [VTG]; plasma sex steroids: 17β-estradiol [E2], 11-ketotestosterone [11-KT] and testosterone [T]; relative gonad [GSI] and liver [HSI] growth; maturation stages of germ cells [MS]; prominence of spawning tubercles [STI]) were investigated. Distinct regional differences in infection rates of bream with L. intestinalis were observed along the Elbe with the highest prevalences at the Czech border (up to 45%) and Magdeburg (up to 65%), areas that are heavily contaminated with complex mixtures of organic chemicals and metals. Parasitized fish of both sexes had significantly lower GSIs and poorly developed gonads (low MS). In males, a significant reduction in the prominence of spawning tubercles occurred. Infected females had significantly lower plasma VTG concentrations. A selective suppression of the sex steroids 11-KT and E2 was observed in male and female bream, respectively. Testosterone was not affected in the same manner in fish of both sexes. At sites with an elevated prevalence of L. intestinalis, the extent of the infection of an individual was significantly correlated with the suppression of the measured biomarkers. However, when applying a linear model to compare regional differences in infection prevalence with biological parameters, not all of the observed differences could be explained by parasitization by L. intestinalis. This indicates that other factors such as pollution may have contributed to the effects on reproductive and endocrine processes that occurred along the river. Given that sites with high prevalences of L. intestinalis were also characterized by elevated pollution, it is possible that there exists a combinatory effect of both pollution and parasitization that can have a serious impact on the reproductive capacity of a population, such as was observed at the sampling site at Magdeburg.

Long-term exposure to continuous light inhibits precocity in European male sea bass ( Dicentrarchus labrax, L.): hormonal aspects

The effect of long-term exposure to continuous light on the hormonal aspects of the reproductive axis was investigated in juvenile male sea bass ( Dicentrarchus labrax, L.) during the first annual cycle. Four-month-old fish were exposed to a simulated natural photoperiod (NP) and a continuous light (24 h) regime (LL) under natural conditions of temperature (13.3–25.8 °C). A dot-blot technique was used to analyse gonadotropin (the common glycoprotein α, GPα; the follicle stimulating hormone beta, FSHβ; and the luteinizing hormone beta, LHβ, subunits) mRNA levels in the pituitary during the experiment. A homologous ELISA was used to determine pituitary sea bream gonadotropin-releasing hormone (sbGnRH) and LH and plasma LH levels; gonadal and plasma sex steroids concentrations were determined by specific immunoassays. LL significantly inhibited the expression of all three gonadotropins subunits in the pituitary. However, no significant differences on plasma LH levels were observed between NP and LL groups throughout the period of the experience. Long-term exposure to LL regime was extremely effective in inhibiting gonadal growth and hence precocious maturation as well as the accumulation of Testosterone (T) and 11-ketotestosterone (11-KT) in the gonads compared to the control group. 11-KT plasma levels remained low and unchanged in the LL group during the study. This work describe important alterations of the endocrine system, particularly at the pituitary–gonad axis provoked by exposure to continuous illumination and discusses the mechanism by which precocity in male sea bass is generated.

Reproductive Responses of Common Carp (Cyprinus carpio) Exposed in Cages to Influent of the Las Vegas Wash in Lake Mead, Nevada, from Late Winter to Early Spring

The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent, nonpotable shallow groundwater seepage, and runoff from the urbanized Las Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To investigate the potential for contaminants in LW influent to produce effects indicative of endocrine disruption in vivo, adult male and female common carp (Cyprinus carpio) were exposed in cages for 42-48 d at four sites in Lake Mead: LW, LX, and two reference locations in the lake. End points examined included gonadosomatic index; gonad histology; concentrations of plasma vitellogenin (VTG) and plasma sex steroids (17beta-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT)); plasma estrogen:androgen ratios (E2:T, E2:11-KT), in vitro production of T by gonad tissue, and hepatopancreas ethoxyresorufin O-deethylase activity. Few differences among fish caged at different sites were potentially attributable to exposure to contaminants

Multigenerational exposure to phytosterols in the mouse

Due to beneficial health effects phytosterols (PS) are increasingly added to functional foods. The aim of the present study was to investigate the chronic effects of a dietary PS mixture (5 mg/kg/day), containing mainly β-sitosterol, on the reproduction of the mouse. General reproductive parameters, postnatal development, growth and survival of pups, weight of sex organs, the concentrations of plasma sex steroids and testicular testosterone were monitored across five generations (F 0–F 4). PS exposure increased the plasma levels of testosterone and decreased the relative uterine weights in the pups of F 2 and F 4 generations. Furthermore, PS exposure increased the concentrations of plasma estradiol in the female pups of F 3 generation. PS supplement also increased the testicular levels of testosterone in the male pups of F 2 generation. In spite of these transitory changes, PS exposure caused no permanent deleterious effects on the reproduction of the mouse.

Plasma sex steroid concentrations and gonadal aromatase activities in African clawed frogs (Xenopus laevis) from South Africa.

Adult African clawed frogs (Xenopus laevis) were collected from a corn-growing region (CGR) and a non-corn-growing region (NCGR) with different exposure profiles for atrazine and related triazines. Physical, chemical, and biological parameters from the catchment areas were also measured. Frogs were surveyed for possible effects of exposure to triazine herbicides on plasma testosterone (T) and estradiol (E2) titers, gonadal aromatase activity, and gonad growth (GSI). Concentrations of both T and E2 varied among locations and were correlated to some accessory factors, such as pH, several ions, and metals. Greatest median plasma T concentrations (males: 19 ng/ml; females: 16 ng/ml) occurred in frogs inhabiting NCGR as compared to those from the CGR (males: 4 ng/ml; females: 1 ng/ml). Median E2 concentrations were also greater in frogs collected from the NCGR (males: 3 ng/ml; females: 28 ng/ml) than those in frogs from the CGR (males: 2 ng/ml; females: 5 ng/ml). Because some exposure to agricultural chemicals at both regions occurred, as did simultaneous exposures to multiple chemicals, a regression analysis was employed. Negative correlations were observed between plasma T concentrations and concentrations of atrazine, deisopropylatrazine, deethylatrazine, and tertbuthylazine in females and between T and diaminochlorotriazine in males. Estradiol in females exhibited a significant negative correlation with atrazine and deethylatrazine. No correlations were observed between gonadal aromatase activity or GSI and any of the agricultural chemicals measured. Median aromatase activities in ovaries varied among sampling sites ranging from 7 to >3000 times greater than those in males when measurable. Testicular aromatase activity was below the detection limit of the assay in male frogs at most of the sites. Although exposure to agricultural inputs did not affect aromatase activities, effects of atrazine or coapplied pesticides on sex steroid homeostasis cannot be excluded at this point.

Diminished aromatase immunoreactivity in the hypothalamus, but not in the basal forebrain nuclei in Alzheimer’s disease

In previous studies we have shown in Alzheimer’s disease (AD) an enhanced nuclear estrogen receptor (ER) α expression in the cholinergic basal forebrain nuclei, i.e. the vertical limb of the diagonal band of Broca (VDB) and the nucleus basalis of Meynert (NBM), and in a number of hypothalamic nuclei, i.e. the supraoptic nucleus (SON), the infundibular nucleus (INF), the medial mamillary nucleus (MMN). We aimed at determining whether the increase in nuclear ERα seen in AD patients was related to a rise in local production of estrogens by aromatase (P-450arom), which is a key enzyme that catalyzes the biosynthesis of estrogens from precursor aromatizable androgens. We confirmed for the first time the presence of aromatase mRNA in neurons and glial cells in the human NBM and the tuberomamillary nucleus by RT–QPCR using laser microdissection. Enhanced aromatase immunoreactivity (ir) was indeed observed in the NBM in AD. However, in contrast a decreased aromatase-ir was found in the SON, INF and MMN of AD patients. In addition, P-450arom-ir was clearly diminished in ependymal and choroid plexus cells in AD. While an increase in aromatase-ir was found in the NBM and SON during normal aging, a decrease in staining was observed in the MMN. No sex differences in young control, elderly control or AD patients were present in any of the nuclei studied. In conclusion, brain P-450arom-ir and the relationship of its regulation with plasma sex steroid levels, estrogen and androgen receptors in the human hypothalamus and basal forebrain are region-specific.

Variation in sex steroids and phallus size in juvenile American alligators ( Alligator mississippiensis) collected from 3 sites within the Kissimmee-Everglades drainage in Florida (USA)

This 3-year study was designed to examine variation in plasma sex steroids, phallus size, and the standard error (S.E.) associated with these endpoints in juvenile alligators collected from 3 sites within the Kissimmee-Everglades drainage (Florida, USA) with varying concentrations of sediment organochlorine contaminants. We hypothesized that decreased plasma sex steroid concentrations and phallus size would be observed in the higher contaminant site when compared to the intermediate and lower contaminant sites. Furthermore, we hypothesized that greater S.E. associated with these endpoints would be observed for the populations from more contaminated sites. We found that differences existed with females from the higher contaminant site exhibiting lower plasma estradiol-17β (E 2) and testosterone (T) concentrations. Males from the higher contaminant site exhibited smaller phallus sizes than males from the intermediate and lower contaminant sites. Smaller phallus size in this case differed from that reported in Lake Apopka male alligators [Gen. Comp. Endocrinol. 116 (1999) 356] in that a significant positive relationship between body size and phallus size existed. No difference among sites was observed in plasma T for males. Lower S.E. was associated with E 2 and T concentrations in females from the higher contaminant site and in phallus size in males from the higher contaminant site. This pattern was opposite to what we had hypothesized. We concluded that variation in plasma E 2 and T concentrations, phallus size, and the S.E. associated with these endpoints exists among the 3 sites with the patterns matching the patterns of organochlorine contamination, although S.E. patterns were opposite to what was predicted.

Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish ( Carassius auratus)

Sexually mature goldfish ( Carassius auratus) of both sexes were exposed to two doses (100 and 1000 μg/l) of the widely used herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino- s-triazine) for a period of 21 days and effects on the concentrations of gonad and plasma sex steroids (testosterone (T), 17β-estradiol (E2) and 11-ketotestosterone (11-KT)), plasma vitellogenin (VTG) and gonad histo-morphology assessed. Atrazine did not show any obvious estrogenic effect in males, as determined by a lack of vitellogenin induction. There were, however, effects of atrazine on plasma androgen concentrations (androgen dynamics) and tissue (plasma and gonad) estrogen concentrations in male goldfish; exposure to 1000 μg/l atrazine induced suppression in both testosterone and 11-ketotestosterone, and resulted in elevated 17β-estradiol, after 21 day of exposure. Further, these suppressive effects on plasma androgens and the induction in estrogen were dose- and time-related. The highest atrazine exposure dose induced structural disruption in the testis and both 100 and 1000 μg/l induced elevated levels of atresia in ovaries.

Amphibians as a model for studying xenoestrogen‐dependent neuroedocrine disruption

Several environmental chemicals are known to interact with functions related with the neuroendocrine system, and many of the endocrine disruptors present in the aquatic environment have estrogenic activity and can be responsible for feminization processes in wild fish and amphibians. The estrogenic effects of such disruptors include several levels of investigation: biological significance ‘in vivo’, and ‘in vitro’ by use of estrogenic biomarkers, and effects on the brain‐pituitary‐gonadal axis. Amphibians spend a great part of their life in the water, and can be considered ‘sentinel species'; therefore, the effects of an estrogenic compound, 4‐nonylphenol, were studied by ‘in vivo’ and ‘in vitro’ experiments. As a biomarker of estrogenic effects, vitellogenin changes were assayed in male frogs and newts; the plasma sex steroid trend was evaluated, and brain aromatase‐like‐immunoreactivity was described. 4‐Nonylphenol treatment was able to induce plasma vitellogenin in male frogs and newts, changes in the secondary sexual characteristics, and increase of androgen plasma levels in the treated male newts. Moreover, a reduction in both the number of ARO‐LI containing neurons and their immunola‐beling intensity was seemingly observed in the more caudal cluster of aromatase containing cells or ventral infundibular nucleus. In conclusion, 4‐nonylphenol seems to affect different targets variously involved in amphibian reproduction.

Seasonal variation in plasma sex steroid concentrations in juvenile American alligators

Seasonal variation in plasma sex steroid concentrations is common in mature vertebrates, and is occasionally seen in juvenile animals. In this study, we examine the seasonal pattern of sex hormone concentration in juvenile American alligators ( Alligator mississippiensis) and make a limited comparison of these seasonal patterns on two different lakes in Florida. Male juvenile alligators from a reference lake, Lake Woodruff, displayed temporal patterns in plasma testosterone (T) concentrations that appear to be seasonal. A similar pattern in plasma estradiol-17β (E 2) was observed in juvenile females from Lake Woodruff. Males had significantly elevated T concentrations during the spring and late summer, whereas females had elevated E 2 in the spring and late summer and significantly depressed E 2 concentrations during the winter. A limited 4-month survey of animals from contaminated Lake Apopka found a lack of such seasonality. These results suggest that: (1) healthy wild populations of juvenile alligators have a prolonged peripubescent period that is marked by seasonal hormonal cycles, (2) juvenile alligators exposed to environmental contaminants can lack such seasonal cyclicity, and (3) future studies of juvenile alligators should incorporate such seasonality into the experimental design.

Plasma sex steroid and vitellogenin profiles during gonad development in wild Mediterranean amberjack ( Seriola dumerilii )

Mediterranean amberjacks, Seriola dumerilii Risso, were caught off the Pelagie Islands, in the south Mediterranean Sea, between May 1997 and June 1999. Fish blood was sampled, and gonads were collected at 10-day intervals throughout the spawning period and at monthly intervals during the resting period. Concentrations of plasma estradiol-17s (E2), testosterone (T), 17,20s-dihydroxy-4-pregnen-3-one (DHP) and vitellogenin (Vtg) in females; and plasma T and 11-ketoT (11-KT) in males were correlated with changes in gonadal development. The first females that had already ovulated (F5) were found in late May. Most mature females (F4) were caught in June. Post-spawned females (F6) were found from late July until September. Estradiol-17s was at baseline levels (<0.02 ng ml−1) during autumn/winter and rapidly peaked (6.29±0.68 ng ml−1) from May to early July. Plasma T levels showed a similar profile and were positively correlated to E2 (r 2=0.668) during the spawning period. Continuously elevated levels of E2 and T were observed during the spawning season in vitellogenic females (stages F3, F4 and F5). Resting females were found during the autumn/winter months. Vitellogenin levels increased during May and peaked in June, reverting to undetectable levels in August, in parallel with sex steroid changes. Plasma DHP levels peaked in June (283.45±97.3 pg ml−1), falling to basal values (<5 pg ml−1) in August. DHP levels were higher in mature females (F4) than in maturing (F3) and in partially ovulated (F5) females. DHP values increased during germinal vesicle migration, peaked during germinal vesicle breakdown and decreased again during complete oocyte hydration. In males, changes in T and 11-KT plasma levels were related to testis development. The highest levels of T (5.76±2.64 ng ml−1) were measured during spermatogenesis and highest 11-KT (5.28±3.6 ng ml−1) in males with milt, from May to June. This study provides information, for the first time, on the relation between plasma sex steroid profiles and gonad development in wild Mediterranean amberjack, a useful benchmark for broodstock monitoring under controlled conditions.

Early identification of sex in cultured white sturgeon, Acipenser transmontanus, using plasma steroid levels

In sturgeon aquaculture, it is advantageous to sex fish as early as possible so that males can be used for meat production and females reserved for caviar production. Currently, growers wait 3 to 4 years before fish are sexed by an invasive surgical examination of the gonads. The goal of this study was to determine when sex could be determined by examining levels of plasma steroids. Populations of white sturgeon were held at three locations and water temperatures (Oregon—13±0.1 °C, Idaho—15±0.1 °C and California—18±0.5 °C). Blood was sampled every 3 months and plasma testosterone (T), 11-ketotestosterone (KT) and estradiol (E2) concentrations were measured by radioimmunoassay (RIA). By 18 months of age, males from California had significantly higher levels of T and KT than females. By 21 months, all males could be separated from females based on T and KT content (>2 ng/ml). However, males and females from Idaho and Oregon had similar levels of T and KT up to 36 and 34 months of age, respectively, and sex could not be delineated based on plasma sex steroid content. There were no differences in E2 levels between males and females in all populations, and at all sample periods. Histological analysis of gonads revealed that all but one of the males from California had well differentiated testes with spermatogonia undergoing mitosis in cysts (stage 2), whereas the testes of all fish from Idaho and Oregon had just begun to differentiate (stage 1). Growth rates of fish were highest in California, intermediate in Idaho and lowest in Oregon. It appears that proliferation of spermatogonia is necessary for production of androgens and that growth rate, as influenced by water temperature, is the main contributor to the early onset of this process. This study has developed a less invasive technique for ascertaining sex in white sturgeon that can be used 1 to 2 years earlier than current methodologies.

Influence of gossypol from dietary cottonseed meal on haematology, reproductive steroids and tissue gossypol enantiomer concentrations in male rainbow trout (Oncorhynchus mykiss )

Rainbow trout (Oncorhynchus mykiss) males were fed, during a 9-month period, five experimental diets where fishmeal proteins were gradually replaced with cottonseed meal (CS) proteins (0, 25, 50, 75 and 100%; diets 1–5, respectively). This study was carried out to evaluate the action and tissue concentrations of gossypol. Growth performance of fish was not affected with the increasing levels of CS in the diets. Haemoglobin concentration and haematocrit significantly decreased in fish fed 100% CS compared with other dietary treatments. Gonadosomatic index, plasma sex steroids (testosterone, 11-ketotestosterone and 17,20β-dihydroxy-4-pregnen-3-one) and sperm characteristics (concentration, motility, protein concentration and lactate dehydrogenase activity) were not negatively affected by increasing levels of CS. For the first time, a comprehensive study of gossypol concentrations, total and (+) and (−)-enantiomers, in several tissues (liver, blood plasma, spermatozoa and seminal plasma) was performed. The concentrations of both (+) and (−)-enantiomers significantly increased with increasing levels of CS in the diet. The highest concentrations were found in the liver of fish fed diet 5 (185 ± 18 μg g−1). In blood plasma, the concentrations of total gossypol were 10 times lower than the one found in the liver, but 10 and 100 times higher than the concentrations in the spermatozoa and seminal plasma, respectively. In all tissues studied, the concentration of (+)-enantiomer was higher than the (−)-enantiomer. The ratio (−)-enantiomer/total gossypol did not change significantly with the increasing levels of CS in the diet. The results of the present study indicate that CS can be used over a period of 9 months to replace fishmeal proteins completely without compromising growth and reproduction of rainbow trout males.