Miroslawa Sokolowska-Mikolajczyk

Di-(2-ethylhexyl)-phthalate disrupts pituitary and testicular hormonal functions to reduce sperm quality in mature goldfish

Di-(2-ethylhexyl) phthalate (DEHP) interferes with male reproductive endocrine system in mammals, however its effects on fish reproduction are largely unknown. We evaluated sperm quality and investigated reproductive endocrine system in mature goldfish (Carassius auratus) exposed to nominal 1, 10, and 100μg/L DEHP. To examine DEHP estrogenic activity, one group of goldfish was exposed to 17β-estradiol (5μg/L E2) for comparison. Following 30d of exposure, sperm production was decreased and suppressed in DEHP and E2 treated goldfish, respectively. Sperm motility and velocity were decreased in goldfish exposed to 100 and 10μg/L DEHP at 15s post-sperm activation, respectively. Compared to control, 11-ketotestosterone (11-KT) levels were decreased at 10 and 1μg/L DEHP at day 15 and 30, respectively. In E2 treated goldfish, 11-KT levels were decreased compared to control during the period of exposure. E2 levels were increased in goldfish exposed to E2, but remained unchanged in DEHP treated goldfish during the period of exposure. StAR mRNA levels encoding regulator of cholesterol transfer to steroidogenesis were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. Luteinizing hormone (LH) levels were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the experimental period. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. The mRNA levels of genes encoding testicular LH and androgen receptors remained unchanged in DEHP and E2 treated goldfish. In contrast to E2 treated goldfish, vitellogenin production was not induced in DEHP treated goldfish and mRNA levels of genes with products mediating estrogenic effects remained unchanged or decreased. In conclusion, DEHP interferes with testis and pituitary hormonal functions to reduce sperm quality in goldfish and does not exhibit estrogenic activity.

Attempts to induce an LH surge and ovulation in common carp (Cyprinus carpio L.) by differential application of a potent GnRH analogue, azagly-nafarelin, under laboratory, commercial hatchery, and natural conditions

Abstract The study was performed in order to try to provoke the gonadotropin wave and ovulation in common carp using a potent GnRH analogue, azagly-nafarelin (AZAGLY), without the use of dopamine (DA) antagonists. Different doses, routes of delivery, and injection protocols were applied, as well as different experimental conditions (laboratory, hatchery, and natural). It has been shown that the best and the simplest way of AZAGLY administration was by intraperitoneal injection. Despite dose (40 or 80 μg/kg body weight) and number of injections (one or two), maximal plasma carp-luteinizing hormone (cLH) concentrations never exceeded 40–50 ng/ml. There was no priming effect on final plasma cLH concentrations. Using different treatment protocols, it was possible to obtain 40–60% ovulated females in laboratory as well as hatchery conditions. However, time of ovulation after AZAGLY treatment in some cases was less compressed in comparison with Ovopel-treated (a local commercial product, containing a GnRH analogue plus the dopamine antagonist, metoclopramide) fish. Egg quality was not altered by AZAGLY treatment. With natural spawning, in unfavourable thermal conditions, AZAGLY treatment induced spawning in a limited number of females. It is postulated that, if antidopaminergic drugs are ever banned from use in aquaculture, AZAGLY treatment alone can be considered as an alternative method of inducing ovulation in cyprinid fish for artificial spawning.

Alternations in neuroendocrine and endocrine regulation of reproduction in male goldfish (Carassius auratus) following an acute and chronic exposure to vinclozolin, in vivo

The fungicide vinclozolin (VZ) is in use globally and known to disrupt reproductive function in male. The present study tested the hypothesis that VZ disrupts testicular function in goldfish (Carassius auratus) by affecting brain-pituitary-testis axis. Goldfish were exposed to 100, 400 and 800 μg/L VZ and 5 μg/L 17β-estradiol (E2) for comparison. In VZ treated goldfish, 11-ketotesteosterone (11-KT) secretion was changed depending on dose and duration period of treatment. Following 7 days of exposure, 11-KT was decreased in goldfish exposed to 800 μg/L VZ, while it was increased in goldfish exposed to 100 μg/L VZ after 30 days of exposure. Circulating E2 level was unchanged in VZ treated goldfish, however the E2/11-KT ratio was increased in a concentration-related manner. In E2 treated goldfish, circulatory 11-KT and E2 levels were decreased and increased, respectively, which resulted in an increase in the E2/11-KT ratio. Exposure to VZ at 100 μg/L caused a significant increase in the circulatory luteinizing hormone (LH) after 30 days. In E2 treated fish circulatory LH was decreased, significantly. Transcripts of genes encoding gonadotropin-releasing hormone and androgen receptor in the brain, and those of genes encoding LH and follicle-stimulating hormone receptors, StAR, CYP17, and 3β-HSD in the testis changed in VZ-treated goldfish depending on concentration and period of treatment. mRNA of genes encoding vitellogenin and estrogen receptor in the liver and cytochrome P450 aromatase in the brain were increased in E2-treated goldfish. The results suggest that VZ-induced changes in 11-KT were due to disruption in brain-pituitary-testis axis and provide integrated characterization of VZ-related reproductive disorders in male fish.

The effects of ghrelin on the in vitro spontaneous and sGnRH-A stimulated luteinizing hormone (LH) release from the pituitary cells of common carp (Cyprinus carpio L.).

In fish, like in mammals, ghrelin affects gonadotropin release acting at the level of the hypothalamus as well as directly on the pituitary gland. In the present study, enzymatically dispersed pituitary cells obtained from sexually mature male and female carp (Cyprinus carpio L.) were incubated in the presence of human ghrelin at the concentration of 10(-7) or 10(-6) M, salmon GnRH analogue (Des-Gly(10), D-Arg(6), Trp(7), Leu(8), Pro(9))-LHRH (sGnRH-A) at the concentration of 10(-8) M or the combination of ghrelin (both concentrations) and sGnRH-A. ELISA method was used for carp LH levels determination in the media collected after 10 or 24 h of incubation. Ghrelin at the concentration of 10(-6) M caused the increase of the spontaneous LH secretion from female pituitary cells only. The combination of ghrelin (both concentrations) with sGnRH-A resulted in the significant elevation of LH levels in the incubations of both male and female pituitary cells in comparison with control incubations as well as with sGnRH-A alone treated cells. The results obtained in this study show that ghrelin functions as LH-stimulating hormone in common carp and that it acts directly on gonadotrophic cells, potentiating also the action of GnRH.

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

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

Seasonal short-term effects of naltrexone on LH secretion in male carp (Cyprinus carpio L.)

In order to evaluate the influence of the season (the stage of gonad maturity) on the modulatory role of endogenous opioid peptides in LH secretion in fish, sexually mature male carp (Cyprinus carpio L.) were intravenously injected with naltrexone-opioid receptor antagonist (5 or 50 microg kg(-1)) in the period of natural spawning (June) or gonad recrudescence (December). Moreover, the possible involvement of the dopaminergic system was studied in fish pre-treated with pimozide (dopamine receptor antagonist) and in intact fish. Blood samples were taken every minute, up to 10 min after naltrexone injection. In June, naltrexone significantly lowered LH levels in comparison to saline injected males. In December, there were no differences between saline and naltrexone-injected carps. In fish pre-treated with pimozide, neither in June nor in December were any significant differences in LH levels between control group and the groups injected with naltrexone found. The results showed that, in male carp, LH secretion under the influence of naltrexone depends on the stage of gonad maturity what suggests that the feedback of gonadal steroids on LH release could be mediated by the endogenous opioids. The role of dopamine in these processes is also discussed.

Dopamine control of LH release in the tench (Tinca tinca).

Tench (Tinca tinca) is apparently the only known member of the Cyprinidae in which ovulation is stimulated following administration of a low dose of GnRH analogue (GnRHa) without a dopamine inhibitor. This study evaluated LH release effectiveness of the most commonly used GnRHa and clarified whether LH secretion followed by ovulation is subject to inhibitory dopaminergic control in tench. Fish were intraperitoneally injected with three types of GnRHa, GnRHa with dopamine inhibitor metoclopramide (combined treatment), or the dopamine inhibitor metoclopramide alone. LH concentrations at five sampling times (0, 6, 12, 24, and 33 h) together with ovulation success and fecundity index were recorded. The combined treatment triggered an almost immediate LH release peak with a gradual decline, and resulted in a high ovulation rate. In contrast to the combined treatment, an application of GnRHa alone at 10 μg kg(-1) induced gradual increase of LH concentrations with peaks close to ovulation time, and with high ovulation success. Significant differences in LH concentrations at 6 and 12h and no differences in ovulation success were found between the combined and the GnRHa alone treatments. Metoclopramide alone induced a small increase in LH with no ovulation. The study presents clear evidence of dopaminergic control of LH release in tench, with a high ovulation rate obtained after application of GnRHa alone or in combination with dopamine inhibitor.

The effects of naltrexone, an opioid receptor antagonist, on plasma LH levels in common carp (Cyprinus carpio L.)

Naltrexone-an opioid receptor antagonist, was administered intraperitoneally to sexually mature male and female common carp in the prespawning period, in order to investigate its effects on spontaneous or sGnRH-A-stimulated LH secretion. Naltrexone and sGnRH-A were injected at the same time. The possible involvement of a dopaminergic system in this process was studied in males pre-treated with pimozide (a dopamine receptor antagonist) 12 h before naltrexone and/or sGnRH-A administration. Blood samples for the analysis of carp LH concentrations were taken just before the injections and then after the injections, serial sampling during 24 h was performed. In male carp, naltrexone (500 or 5000 microg kg(-1)) decreased spontaneous LH release, but there were no effects of naltrexone on sGnRH-A-stimulated LH secretion. In males pre-treated with pimozide, a similar response to naltrexone injection (500 microg kg(-1)) as in pirnozide non-treated fish, was observed. The highest dose of naltrexone, 5000 microg kg(-1), significantly stimulated LH release, in response to sGnRH-A administration in pimozide pre-treated males. In female carp, contrary to males, naltrexone at a dose of 500 microg kg(-1), caused significant stimulation of spontaneous LH release. These data indicate that endogenous opioid peptides modify LH secretion in sexually mature carp. In males, they stimulate LH secretion, acting rather on the hypothalamic GnRH system and in females, opioids inhibit LH release by the influence on the dopaminergic system.

The effect of polychlorinated biphenyls mixture (Aroclor 1254) on the embryonic development and hatching of Prussian carp, Carassius gibelio, and common carp, Cyprinus carpio (Actinopterygii: Cypriniformes: Cyprinidae)

Background. Polychlorinated biphenyls (PCBs) are persistent and bioacumulative chemical pollutants which exert negative physiological effects o n the reproductive system of mature male and female fish. PCBs present in the aquatic environment may also have an influence on developing embryos. The aim of this study was to inves- tigate the effect of Aroclor 1254, a polychlorinated biphenyls mixture, on the embryonic development of Prussian carp, Carassius gibelio (Bloch, 1782), and common carp, Cyprinus carpio L. Materials and methods. The samples of eggs obtained from 4 females of each species were divided into t wo dishes and incubated separately after fertilization (common carp) or activation (Prussian carp) with common carp sperm. The incubation, with Aroclor 1254 (1 or 10 ng · mL -1 ) or in water with no PCB added, lasted for 4 days. The mortality, hatching rate, number of hatched larvae, and number of deformed larvae were observed. Results. After 24 h of incubation of Prussian carp and common carp eggs there were no significant differences in the percentage of living eggs between t he Aroclor 1254 treated groups and the control one. The lowest tested concentration of PCB (1 ng · mL -1 ) accelerated the hatching of Prussian carp larvae at 75 h of incubation. The significant increase in the percentage of deformed larvae was observed only in the experiment with common carp eggs incubated with Aroclor 1254 at the concentration of 10 ng · mL -1 . Conclusion. Results of the presented data showed that Aroclor 1254 (at tested concentrations) is not harmful for the development of activated Prussian carp eggs but teratogenic effect was observed in the case of common carp embryos. K eywords: PCBs mixture, common carp, Prussian carp, embryonic development, reproduction

Differential impact of recombinant human activin on LH secretion in male and female common carp during wintering and spawning period

The pituitaries of male and female common carps were subjected to the treatment with recombinant human activin A. Basal LH secretion decreased in females during wintering, but stimulated basal and sGnRH-induced LH secretion during ovulatory period. It stimulated LH secretion in males during wintering, but not during spawning, suggesting sex-specific effect of activin on LH secretion in common carp.