Variation in the reproductive potential of Schistocephalus infected male sticklebacks is associated with 11-ketotestosterone titre

Parasites can impact host reproduction by interfering with host endocrine systems, but the adaptive nature of such effects is disputed. Schistocephalus solidus plerocercoids are parasites of three-spined sticklebacks Gasterosteus aculeatus that are often associated with impaired host reproduction. Here, we relate reproductive behavior and physiology to levels of the androgen 11-ketotestosterone (11KT) in naturally infected and non-infected male sticklebacks from two UK populations. In one population infected males harbored heavy infections and showed uniformly reduced 11KT titres and kidney spiggin (nesting glue protein) content compared to non-infected fish. However in a second population infection levels were more variable and males with smaller infections recorded 11KT and spiggin titres that overlapped those of non-infected fish; among infected males from this population 11KT and kidney spiggin also both correlated negatively with infection severity. Male reproductive behavior correlated closely with 11KT titre in both populations, and infected males with high 11KT levels exhibited normal reproductive behavior. Our results suggest that Schistocephalus infection per se does not block reproductive development in male sticklebacks, and that some male fish may have the ability to breed whilst infected. Our results are not consistent with the hypothesis that Schistocephalus adaptively castrates male hosts via endocrine disruption; rather they support the hypothesis that reproductive disruption is a side effect of the energetic costs of infection.     

Androgen and behavior in the male three-spined stickleback,Gasterosteus aculeatus I.--changes in 11-ketotestosterone levels during the nesting cycle

Circulating 11-ketotestosterone (11KT) levels are higher during the courtship phase than during the later parental phase in a number of male teleosts. The present study describes the temporal changes in 11KT levels and their relationships to changes in courtship behavior, after different number of spawnings, over the nesting cycle of the male three-spined stickleback, Gasterosteus aculeatus, a small teleost showing pronounced paternal behavior. Plasma 11KT levels, measured by radioimmunoassay, were approximately 34 times higher during the initial courtship phase than at the end of the following parental phase in spawned males. In addition, males that spawned with three or more females on a single day showed an earlier decline in 11KT levels and in courtship behavior compared to males that were only allowed to spawn with a single female. Plasma 11KT levels remained high in males not allowed to spawn.     

Effects of fasting on temporal changes in plasma concentrations of sex steroids, growth hormone and insulin‐like growth factor I, and reproductive investment in Arctic charr

Proportions of maturing fish and reproductive output [egg size, relative fecundity, spermatocrit and gonado‐somatic index ( I _G)] were studied in repeat‐spawning (+4 year old) male and female Arctic charr Salvelinus alpinus , subjected to periods of fasting. Groups of individually tagged Arctic charr were fasted for c . 3, 6, 7 and 9 months, from November 1998. In the period February to November 1999, size (fork length and mass), specific growth rate ( G ), condition factor ( K ) and plasma concentrations of growth hormone (GH), insulin‐like growth factor I (IGF‐I), oestradiol‐17β(E₂; females) and 11‐ketotestosterone (11KT; males), were monitored monthly. Maturing fish in each group started to gain mass soon after food was made available, and both sexes reached the highest K and G c . 2 months after the onset of feeding. The fasting regimes resulted in different growth patterns during spring and summer when energy stores are normally replenished in Arctic charr, and K prior to the breeding season was significantly higher in the groups fasted for 3 and 6 months compared to the groups fasted for 7 and 9 months. There were significant positive correlations between K during the period prior to the breeding season and reproductive output in terms of the I _G, spermatocrit and relative fecundity. There was, however, no clear relationship between the length of starvation and the proportion of maturing fish. Likewise, no clear relationships were found between reproductive development and plasma levels of GH and IGF‐I, although both showed marked seasonal changes, being 'down‐regulated' during winter months and 'up‐regulated' throughout summer months.     

Stress-induced changes in concentrations of plasma sex steroids in black bream

Cortisol levels of black bream Acanthopagrus butcheri at capture did not change with time of day, gonadal stage or season and were 1·9±0·2 and 2·8±0·4 ng ml⁻¹ for male and female fish, respectively. Confinement resulted in significantly elevated cortisol levels at all time periods; however, levels after 24 h of confinement were significantly lower than peak cortisol levels (15 min for males and 1 h for females). Confinement stress resulted in reduced levels of 17β-oestradiol (E2) and testosterone (T) within 1 h in sexually mature females. In mature males, suppression of T and 11-ketotestosterone (11KT) occurred after 30 min and 6 h of confinement, respectively. The relationship between confinement stress and levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β P) was more complex, with levels in males being elevated after 15 min and 24 h and suppressed after 6 h of confinement. In contrast, 17, 20β P levels in females were elevated after 1 h of confinement. In regressed females, plasma E₂ and T concentrations were low at capture and were not affected by confinement stress whereas plasma 17, 20β P was elevated within 1 h. This study indicates that stress exerts a rapid inhibitory effect on gonadal steroidogenesis.     

Serum levels of testosterone, 11‐ketotestosterone and oestradiol‐17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment

Changes in serum concentrations of two androgens, testosterone (T) and 11‐ketotestosterone (11KT), and oestradiol‐17β(E2) in male and female giant sturgeon Huso huso , Russian sturgeon Acipenser gueldenstaedtii and stellate sturgeon Acipenser stellatus were studied at different stages of gonadal maturity and after final maturation induced by hormonal treatment. Both male and female fish displayed a distinct increase in serum steroid concentrations during gonadal development. 11KT levels were significantly higher in males than females, with a positive correlation detected between 11KT and T concentrations. In maturing males and females, higher values of both 11KT and T were observed in stellate sturgeon compared to giant and Russian sturgeons. Vitellogenesis and high E2 levels were correlated in maturing sturgeon females.     

Sex differences in pectoral muscles but not in pectoral fins in the three‐spined stickleback Gasterosteus aculeatus

The pectoral muscle index ( I _PM)( I _PM = 100 M _PM M⁻¹, where M _PM and M are the pectoral muscle and body masses, respectively) fin‐area and fin ray length were studied over a year in male and female three‐spined stickleback Gasterosteus aculeatus from a marine population (Öresund, Sweden) kept under simulated natural light and temperature conditions. A castration‐replacement experiment was used to test androgen effects on the I _PM, fin‐area and fin ray length. Non‐breeding males were castrated or sham‐operated in winter ( i.e . the fish had low levels of androgens). Castrated control and sham‐operated fish were implanted with empty Silastic capsules and castrated groups with capsules containing the androgens testosterone or 11‐ketoandrostenedione into the abdominal cavity. The experiment was terminated after 41 days, when the controls had matured. No morphological differences were found in pectoral fins between sexes during the year, except during the peak breeding season (May), where females showed larger fin‐area and longer fin ray in length compared to males. No effects of androgens treatment or of castration on pectoral fin‐area or fin ray length was observed. Breeding and non‐breeding males showed higher I _PM compared to females. The lower I _PM in females than in males could not be explained by the larger gonads in the former alone, as a sex difference in I _PM was still present after deduction of the ovaries from the female body mass. The I _PM was higher in sham‐operated compared to castrated fish. No effects of androgens treatment on I _PM was observed.     

Release of sex steroids into the water by roach

Electro‐olfactogram (EOG) recordings of the olfactory epithelium of both male and female roach Rutilus rutilus demonstrated that both sexes were able to detect free and glucuronidated 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) with high sensitivity. Male, but not female, roach were also sensitive to androstenedione. Sexually mature female roach were shown to release free 17,20β‐P, glucuronidated 17,20β‐P and androstenedione into the water; for all three steroids, the rate of release was significantly enhanced by injection of carp pituitary extract (CPE). A series of trials was also carried out which showed that mature males, and to a lesser extent immature males and females, were able also to release free and glucuronidated 17,20β‐P, both before and after CPE treatment. Water extracts from containers that had held CPE‐treated mature male and female roach were examined for the presence of other steroids. This revealed that free and glucuronidated 17,20β‐P plus free and glucuronidated 17,20β,21‐trihydroxy‐4‐pregnen‐3‐one (17,20β, 21‐P) predominated in water extracts from both sexes. The free moieties of 17,20α‐dihydroxy‐4‐pregnen‐3‐one, 17‐hydroxyprogesterone and 11‐deoxycortisol were found at concentrations which were between four and 20 times lower than those of free 17,20β‐P. Androstenedione was found at concentrations which were 25‐fold lower than those of 17,20β‐P. Despite its apparent high rate of release by sexually mature male and female roach, free 17,20β,21‐P was found not to exhibit any EOG activity at the highest dose tested (10⁻⁷ M).     

Androgen and behavior in the male three-spined stickleback,Gasterosteus aculeatus. II. Castration and 11-ketoandrostenedione effects on courtship and parental care during the nesting cycle

Courtship declines and ceases while parental care increases in the presence of developing eggs during the nesting cycle of the male three-spined stickleback, Gasterosteus aculeatus. Furthermore, circulating 11-ketotestosterone (11KT) levels are higher during the initial "courtship phase" than during the later "parental phase," similar to that found in other paternal fishes. This study aimed to investigate a possible functional relationship between changes in 11KT levels and changes in reproductive behavior during the nesting cycle. To this end, groups of nonspawned and spawned male sticklebacks were sham-operated, castrated, or castrated and treated with 11-ketoandrostenedione (11KA), and the effects of the treatments on courtship and parental care were studied. Castration removed circulating 11KT, while 11KA replacement prevented the natural decline in 11KT during the parental phase (11KA converts to 11KT extratesticularly), as assessed by radioimmunoassay. Regardless of treatment, parental care remained low and courtship was present in all nonspawned males, even at the end of the experiment. However, courtship did eventually decline in castrated nonspawned males compared to the other two nonspawned groups. In all treatments of spawned males there was a drastic decline in courtship and an increase in parental care. In castrated spawned males, however, the decline in courtship came earlier than in the other two spawned groups. 11KA treatment did not prevent the natural decline in courtship/increase in parental care in spawned males, indicating that the natural decline in 11KT is not responsible for the main portion of the rapid changes in these behaviors over the stickleback's nesting cycle. The limited effects of castration also exclude other gonadal hormones from being responsible for most of these changes.     

Ontogeny of gonadal sex development relative to growth in fathead minnow

Ovarian differentiation of fathead minnow Pimephales promelas occurred at between 10 and 25 days post‐hatch (dph)(8–11 mm fork length, L _F, and 7–12 mg), and was characterized by the presence of meiotic cells in the centre of the gonad, location of the somatic cells at the periphery of the gonad and the formation of an ovarian cavity. In contrast with the developing ovary, in the presumptive testis somatic cells were scattered throughout the gonads and this was evident from 25 dph (fish >10 mm and >11 mg). In males, at 60 dph (15–26 mm and 39–220 mg) the efferent ducts (sperm ducts) were apparent and the testis lobules started to form, but germ cells (spermatogonia) did not enter meiosis until between 90 and 120 dph. Fish of both sexes reached full sexual maturity at between 120 and 150 dph (males: 33–59 mm and 400–2895 mg; females: 24–48 mm and 160–1464 mg). Differences in body size ( L _F and mass) between males and females were only apparent when the fish were approaching full sexual maturity (120 dph).     

Plasma levels of androgens and cortisol in relation to breeding behavior in parental male bluegill sunfish, Lepomis macrochirus

Like many teleosts, male bluegill sunfish (Lepomis macrochirus) provide sole parental care. To understand some of the proximate costs of parental care, we measured body condition and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and cortisol in nesting bluegill males during pre-spawning, spawning and parental care stages. T and 11KT were at their highest mean levels during the pre-spawning period and decreased to lower levels early during the parental care period before rising again when the eggs hatched. Cortisol levels fluctuated across the breeding stages, but there was a noticeable increase from low levels on the day of spawning during the first 2 days of parental care when egg fanning is most intense. Levels of all hormones varied considerably among males, with androgen levels often correlating positively with a male's body condition. We also demonstrate, using a brood reduction experiment and repeated sampling of known individuals, that the presence of eggs affects hormone levels shortly after eggs hatch. Parental males in better body condition had higher levels of androgens during parental care. Males that were known to renest later in the season also had higher androgen levels and were in better body condition during the first nesting bout than males only known to have nested once. However, circulating levels of cortisol did not differ significantly between these groups. We discuss our findings in the context of proximate and ultimate costs of parental care and propose several reasons why elevated androgen levels may not be as incompatible with the expression of paternal care in male teleosts, as compared with avian and mammalian fathers.     

Reproductive biology of gag in the southern Gulf of Mexico

Aspects of the reproductive biology of gag Mycteroperca microlepis in the southern Gulf of Mexico were studied by following seasonal variations in the gonado‐somatic index and through histological examination of gonads. Gag were collected from inshore and offshore waters of the Campeche Bank, Yucatan, Mexico, between April 1996 and December 2001. This species is a protogynous hermaphrodite, and appeared to be depth‐size distributed. The smallest gag (9–49 cm L _F) collected were all juvenile females, and were caught in inshore waters (1–10 m depth), while the largest (49–116 cm L _F), mainly adult females, males and transitionals, were captured in offshore waters (33–167 m depth). Overall the offshore male to female ratio was female‐biased (1 : 3·3) and differed significantly from unity. The species spawns at depths of c . 50–53 m, from early winter to mid‐spring, with peak spawning activity occurring between January and March. Fifty per cent of females reached first maturity at 72·1 cm L _F. At 103 cm L _F, 50% of sampled females had changed into males. Gag can be considered a monandric species, and sexual transition for this grouper seemed to occur in fish distributed within a narrow size range (85–111 cm L _F). The results are compared with those of other authors for gag stocks from the south‐east Atlantic coast of the U.S.A. and the north‐east Gulf of Mexico.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Pheromone of male blue gourami and its effect on vitellogcnesis, steroidogenesis and gonadotropin cells in pituitary of the female

Female Trichogaster trichopterus were exposed to aquarium water in which males had built nests. Gonadotropin cells in the pituitary gland, and exovitellogenesis and steroidogenesis in the ovary were studied. In females in which the percentage of oocytes in vitellogenesis (%V) was low initially, it rose significantly in comparison with an unexposed control group. In females in which the %V was higher initially, it increased further, and in addition a significant percentage of oocytes reached maturation. Thin layer chromatography, using the precursors ³H-pregnenolone and ¹⁴progesterone, revealed high yields of the steroids 17β-estradiol (E₂), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), 5β-pregane,3α,17α,20β-triol (5β-P-triol) and 11-ketotestosterone (11KT) in both experimental groups. Significant differences were found in E₂, 17,20-P and 5β-P-triol between the test and control groups. The immunoresponse of GtH-producing cells in the pituitary of the females maintained in nest water was lower than in the control group, suggesting that the GtH was secreted from the cells, which would explain the vitellogenic and steroidogenic changes found in the ovary.     

Calcium metabolism and osmoregulation during sexual maturation of river running Atlantic salmon

As Atlantic salmon return from the ocean to undertake the anadromous spawning migration up the river of origin, profound changes in calcium metabolism and osmoregulation take place. Using tartrate resistant acid phosphatase as a marker, scale osteoclast activity was found to increase throughout sexual maturation and spawning migration. Thus, the participation of osteoclasts in the elevated scale resorption observed during this phase is established. As calcium was simultaneously accumulated in the female gonads, it is proposed that the scales are resorbed in order to provide calcium for the growing ovaries. Plasma oestradiol-17 β levels were elevated in females during sexual maturation, and had decreased at the time of spawning. Plasma testosterone levels were similar in males and females during the first part of the upriver migration, but had increased in males and decreased in females at spawning. In addition to the role of these sex steroids in the gonadal growth, their possible involvement in the increased scale resorption during this phase is discussed. Plasma growth hormone and thyroxine levels were elevated in both sexes at spawning, with the triiodothyronine/thyroxine (T₃/T₄) ratio declining sharply, indicating possible roles for these hormones in the maturational process. The relatively low gill Na⁺, K⁺-ATPase activity of salmon caught in the estuary implies that the fish had already adapted to a hypoosmotic environment. During the upriver migration, the gill Na⁺, K⁺-ATPase activity decreased further, indicating that the hypoosmoregulatory ability was suppressed further during sexual maturation and spawning migration.     

The effect of reduced temperature and salinity on the blood physiology of juvenile Atlantic cod

The osmolality and ionic composition of the blood of juvenile Atlantic cod Gadus morhua and their response to conditions of reduced temperature and salinity in summer‐ and winter‐acclimated individuals was investigated. Haematocrit percentage was relatively stable throughout the experimental procedures. Summer‐acclimated juvenile Atlantic cod had higher plasma osmolality than winter‐acclimated fish in ambient conditions. Plasma Na⁺ levels were, however, higher in winter conditions, while Cl⁻ did not vary between seasons. Temperature reduction (12, 9 and 6° C in summer and to 6 and 4° C in winter) induced a significant response in plasma osmolality and Na⁺ levels in summer, but only in Na⁺ levels in winter‐acclimated fish. A pronounced effect was seen in the summer 6° C treatment. Salinity treatments (24, 16 and 8) had a significant effect on almost all the variables in both summer and winter and resulted generally in dilution of ionic and osmotic concentrations of the plasma. This effect was pronounced in the lowest temperature treatments, with the greatest reduction observed in the summer 6° C treatment. This could suggest that winter‐acclimated fish are physiologically adapted to cope with lower seawater temperatures as opposed to summer‐acclimated fish.     

Relationship of plasma steroids to germ cell development and the presence of protamine mRNA in rainbow trout during the induction of spermatogenesis with partially purified salmon gonadotropin

The gonadosomatic index (GSI) of pre–pubertal male rainbow trout, which had been injected biweekly with partially purified salmon gonadotropin (sG–G100, 50 μ mUg kg⁻¹ body weight), increased from 0.05 to 1.85 over 21 weeks from injection, while control GSI remained below 0.05. Plasma testosterone (T) increased from 2 to 11.34 ng ml⁻¹ by week 21 in injected fish, while control level remained below 1.5 ng ml⁻¹. In injected fish plasma 11–ketotestosterone (KT) and 17,20–dihydroxyprogesterone (17α20βP) levels increased from 20.2 to 41.9 and 8.9 to 219.7 ng ml⁻¹ respectively. Plasma T, 11–KT, and 17α20βP were all correlated with the GSI ( P <0–001) in injected fish. The most advanced stage of germ cells present in the control fish were spermatogonia. However, in injected fish spermatozoa were present by week 21. Eggs fertilized at this time with spermatozoa from injected fish achieved a 78% fertilization rate, whereas the testicular homogenate was incapable of fertilizing eggs.     

Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta . During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml⁻¹) and female (plasma VTG levels >800 μg ml⁻¹) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.     

Elevated 11-ketotestosterone during paternal behavior in the Bluebanded goby (Lythrypnus dalli)

The relationship between androgens and paternal behavior is not straightforward, potentially because of the diversity of tasks a male must undertake to maximize reproductive success, notably alternating between courtship, aggression, and offspring care. In some species, these events are separated in time, but in others they are coincident. The endocrine profiles of species that simultaneously court, parent, and defend a nest, such as male bluebanded gobies (Lythrypnus dalli), are not well understood. We sampled a potent fish androgen, 11-ketotestosterone (KT), at different life history stages (experienced parenting males, experienced males not actively parenting, inexperienced males with their first clutch, and females), to examine this relationship. We found that experienced parenting L. dalli males have the highest KT levels of any group, while none of the other groups differed significantly. Males showed elevated KT levels when they had eggs compared to when they did not. Our data suggest that KT facilitates at least some aspects of parental care in L. dalli.     

Puberty and recrudescence in cultured male yellowtail flounder: plasma androgens and spermatogenesis

Two year classes of cultured male yellowtail flounder Limanda ferruginea were followed in order to examine puberty and recrudescence at the gonadal level, and to assess the impacts of culture conditions on male reproduction. Cultured males demonstrated a propensity for maturation as 1 year‐old fish. Pubertal activation of testicular steroid production was associated with the appearance of primary spermatocytes, but a single case of a premeiotic endocrine activation was detected. The onset of puberty occurred during September and October, when adults were post‐spawned or in early recrudescence. Once initiated, pubertal development proceeded in an uninterrupted progression to full maturity by June at 22 months of age. Spermatogenetic activity during the autumn led to a peak in the gonado‐somatic index and total testicular mass by December. Milt could be first expressed from some pubertal males at this time, well in advance of the May to August spawning period in captivity. Meiotic activity could be absent from the testes by the spring, but residual areas of meiotic activity were found in pubertal males of one year class which may indicate a slower spermatogenetic rate. Androgen levels peaked during prespawning and early spawning; 11‐ketotestosterone (11‐KT) was the dominant androgen throughout spermatogenesis. Recrudescing 2 year‐old males showed greater testicular development and androgen production. Spermatogenetic patterns were similar to those seen in pubertal males, with the distinction that recrudescing males had a more discernable mitotic phase prior to meiotic activity; mitotic and meiotic activity overlapped considerably during early puberty. Testicular development well in advance of spawning may confer an evolutionary advantage to males of this cold‐water marine flatfish.