Effects of weathered polyethylene microplastic ingestion on sexual maturation, fecundity and egg quality in maturing broodstock Atlantic cod Gadus morhua.

Microplastics (MPs) have become a global issue as they are omnipresent in the ocean. Fish ingesting MPs through feed could be affected in their physiological function, e.g., disrupted enzyme production and function, reduction of feeding and reproductive failure. This study assessed the effects of feed containing naturally weathered MPs from the Oslofjord (Norway) on the reproductive physiology of Atlantic cod (Gadus morhua). Farmed cod broodstock were fed either control (C-diet) or feeds containing 1% microplastic (MP-diet) starting nine months prior to spawning, from June until May. No major differences were found between diet groups in overall biometrics or gonad histology. Sex steroid levels (testosterone, 11-ketotestosterone and 17ß-estradiol) resulted in expected profiles increasing over time without any significant differences between treatments. Gene expression levels of the steroidogenic enzyme 20ß-hydroxysteroid dehydrogenase (20ß-hsd) and vitellogenin1 (vtg1) showed significant differences between dietary treatments with lower expression in the control group. This can be a direct effect of MPs, but endocrine disrupting effects of potentially leachable plastic additives cannot be completely ruled out. Thus, these enzymes could be indicators of exposure to contaminants that disrupt sexual maturation by affecting the production of primarily maturation-inducing steroid. Although the concentration of MPs employed in this study may not be high enough to elicit any observable short-term biological effects, the observed gene expression suggests that long-term consequences should be considered caused by an expected increase of MPs in marine environments.

Synthesis of 17,20beta,21-trihydroxypregn-4-en-3-one by ovaries of reproductively mature Atlantic cod Gadus morhua.

Atlantic cod Gadus morhua ovaries were incubated in vitro with tritiated 17-hydroxypregn-4-ene-3,20-dione (17-P) to determine whether 17,20beta-dihydroxypregn-4-en-3-one (17,20beta-P) or 17,20beta, 21-trihydroxypregn-4-en-3-one (17,20beta,21-P), or both, were more likely to be the steroid responsible for inducing oocyte final maturation (i.e. resumption of meiosis). Only 17,20beta,21-P was produced, in addition to 11-deoxycortisol (17,21-P), which is intermediate between 17-P and 17,20beta,21-P. Also, the 5beta-reduced forms of 17-P, 17,21-P and 17,20beta,21-P were all found. Some sulphation of 21-hydroxylated steroids was demonstrated. The ability of female G. morhua to make 17,20beta,21-P but not 17,20beta-P was confirmed by radioimmunoassay of plasma samples from spawning fish. Although small amounts of 17,20beta-P immunoreactivity were detected in a few plasma samples, this was shown, by thin-layer chromatography, to be mostly due to cross-reaction with other unidentified compounds. The evidence strongly suggests that 17,20beta,21-P is more likely than 17,20beta-P to be the maturation-inducing steroid in G. morhua.

Effects of the sea louse Lepeophtheirus salmonis on temporal changes in cortisol, sex steroids, growth and reproductive investment in Arctic charr Salvelinus alpinus.

Groups of mature (5+ year old) Arctic charr Salvelinus alpinus held in sea water were exposed for 34 days to either a high (mean +/-s.e. 0.15 +/- 0.01 sea lice Lepeophtheirus salmonis g(-1) fish mass) (HI), medium (0.07 +/- 0.00 sea lice g(-1) fish mass) (MI) or no [control (C)] sea-lice infection during early stages of gonad development (June to July). Infection with sea lice resulted in increased plasma cortisol concentrations and this was related to intensity of infection; females tended to have higher cortisol concentrations than males at high infection intensities (HI group: female c. 130 ng ml(-1); male c. 80 ng ml(-1)). Plasma osmolality (C c. 330, MI c. 350 and HI c. 415 mOsm) and chloride concentrations (C c. 135, MI c. 155 and HI c. 190 mM) increased significantly with infection intensity, indicating osmoregulatory problems in infected fish. A strong positive relationship between plasma osmolality and cortisol concentration was recorded. Plasma sex-steroid concentrations were influenced negatively by sea-lice infection, particularly in the HI group, and were inversely related to plasma cortisol concentrations. The most heavily infected fish postponed the initiation of reproductive development until exposed to fresh water and timing of ovulation tended to be delayed in these fish. Growth rate and condition were negatively influenced by sea-lice infection and growth rate was inversely related to plasma cortisol concentrations. Sea-lice infection resulted in mortality among females in the HI group, and the proportion of maturing females was lower in the MI group (46%) than in the controls (85%). Egg production in the MI and HI groups was c. 50 and 30% of the C group. Egg size, embryonic survival and fry mass did not differ across groups. Sea lice influence reproductive development and egg production in S. alpinus, and consequently these parasites may influence populations via sublethal effects on broodfish, affecting growth and condition, and their reproductive output.

Plasma-sulfated C21-steroids increase during the periovulatory period in female common wolffish and are influenced by temperature during vitellogenesis.

Plasma concentrations of steroids during the periovulatory period were measured in female common wolffish reared at three different temperatures. Steroids were quantified by radioimmunoassay (RIA). Two "broad-spectrum specificity" RIAs-one which detects C21-steroids with a 17,20beta-dihydroxyl configuration (17,20beta-steroids) and the other which detects C21-steroids with a 5beta-reduced, 3alpha-hydroxyl configuration (5beta,3alpha-steroids)-picked up very large amounts of cross-reacting material (1.7 microg ml(-1) in one fish) in the sulfate fraction of plasma from ovulating females. Reverse-phase high-performance liquid chromatography and thin-layer chromatography revealed two major steroids: 5beta-pregnane-3alpha,17,20beta-triol (80%) and 5beta-pregnane-3beta,17,20beta-triol (20%). The sulfated forms of these steroids were elevated 4 to 6 days before and during ovulation, compared with those of females in vitellogenic and postspawning condition, in which concentrations were below 2.0 ng ml(-1). In the three groups of fish held at 4, 8, and 12 degrees C during vitellogenesis, but returned to 4 degrees C just prior to the spawning season, the mean concentrations of sulfated 17,20beta-steroids in ovulating females were 530, 635, and 325 ng ml(-1), respectively. The corresponding concentrations of free 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P; the maturation-inducing steroid in many teleosts) were 0.88, 0.86, and 0.57 ng ml(-1), respectively. Only minute amounts of 17,20beta,21-P and its sulfated derivatives were detected. Significantly lower steroid concentrations in the 12 degrees C group indicate that steroid synthesis and/or metabolism during the periovulatory period are influenced by the temperature experienced during vitellogenesis. In male fish, plasma concentrations of both sulfated 17,20beta-steroids and free 17,20beta-P were low (< 2.0 ng ml(-1)) at all times.

Arctic charr (Salvelinus alpinus) vitellogenin: development and validation of an enzyme-linked immunosorbent assay.

Vitellogenin (Vtg) was isolated from plasma of estradiol-17 beta-treated Arctic charr males by double precipitation with MgCl2-EDTA and distilled water, followed by ion-exchange chromatography. The monomeric form of Vtg, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 158 kDa. The purified Vtg was used to raise a polyclonal antibody for Vtg (AbVtg), and the specificity of the AbVtg was assessed by Western blot analysis. No cross-reactivity was observed with plasma from control males. Using this AbVtg, a competitive enzyme-linked immunosorbent assay was developed. The detection limit of the assay was 2 ng ml-1, and the intra- and inter-assay variations determined from plasma samples were 8.6 and 13.3%, respectively. The assay was validated by quantification of Vtg in plasma samples obtained during a reproductive cycle of Arctic charr. Vtg of females increased gradually from 3 mg ml-1 in early March to a peak value of 22 mg ml-1 in late August, followed by a rapid drop to 2 mg ml-1 at the time of spawning in mid-October. The temporal changes in plasma Vtg of females correlate well with the reproductive cycle. Vtg was undetectable in males, except on some sampling dates during July-September when minute amounts (3-13 micrograms ml-1) were detected in some individuals.