Impact of absolute food deprivation on the reproductive system in male goldfish exposed to sex steroids.

There is a link between metabolism and reproduction as metabolic hormones affect hypothalamus-pituitary-testis (HPT) hormonal functions and vice versa. The aim of the present study was to investigate the effects of negative energy balance on the reproductive system in male goldfish exposed to testosterone (T) and 17ß-estradiol (E2). Following 7 days of food deprivation (FD), ANOVA models showed significant FD × sex steroid interactions on sperm quality and circulating sex steroid levels. When FD effects were investigated, 11-ketotestosterone (11-KT) level and sperm motility and velocity decreased in food-deprived goldfish in the control group. In E2-exposed goldfish, FD decreased sperm production in addition to sperm motility and velocity that coincided with an elevation of circulating E2 level. However, FD did not significantly impact sex steroids and sperm quality in T-exposed goldfish. ANOVA models showed non-significant FD × sex steroid interactions for HSI, GSI, circulating luteinizing hormone (Lh) level, and metabolic (preproghrelin, goat and nucb2) and reproductive (kiss1, gpr54 and gnrh3) mRNAs. Furthermore, results showed that FD decreased HSI, and increased Lh levels and testicular preproghrelin and goat mRNAs, while sex steroids increased mid-brain nucb2, kiss1 and gpr54 mRNAs. Together, our results suggest that FD-induced inhibition of androgenesis resulted in diminished sperm quality associated with activation of the testicular ghrelinergic system, and negative feedback of 11-KT increased Lh level. The FD-induced testicular metabolic and hormonal system was impacted in goldfish exposed to sex steroids. However, the negative effects of FD on sperm quality were accelerated in E2-exposed goldfish due to estrogenic activity. This study provides novel information to better understand metabolic-associated reproductive disorders in fish.

The Effect of [(D-Ala6, Pro9NEt)mGnRH-a + Metoclopramide] (Ovopel) on Propagation Effectiveness of Two Breeding Lines of Common Carp (Cyprinus carpio L.) and on Luteinizing Hormone and 17α,20ß-Dihydroxyprogesterone Levels in Females during Ovulation Induction.

The study evaluates the impact of Ovopel on the reproductive effectiveness of carp from Polish line 6 and Lithuanian line B and the release of luteinizing hormone (LH) and 17α,20ß-dihydroxyprogesterone (17α,20ß-DHP) in females from these lines during ovulation induction. The levels of both hormones were determined in blood plasma samples taken just before the priming injection of Ovopel (0 h), at the time of administering the resolving dose of Ovopel (12 h), and after the next 12 h (24 h). Following Ovopel treatment, the mean egg weight obtained for line 6 was higher, but not statistically different, than that observed for line B. Egg quality, on the other hand, was significantly higher in line B. Female provenance did not significantly affect the number of eggs and living embryos after 70 h incubation. However, the total egg number for line 6 was higher. The mean number of living embryos (70 h) was similar for both lines. LH concentrations at 0, 12, and 24 h were not statistically different between the lines. A comparison of LH concentrations between ovulated and non-ovulated females at different sampling times revealed no significant differences either within or between the lines. Statistically significant differences in LH levels were found for both ovulated and non-ovulated females from a given line between the sampling times. The results for 17α,20ß-DHP were similar, with only one difference: 24 h after the priming dose of Ovopel, 17α,20ß-DHP levels in ovulated fish were significantly higher compared with non-ovulated females, but only in line 6.

A chronic exposure to bisphenol A reduces sperm quality in goldfish associated with increases in kiss2, gpr54, and gnrh3 mRNA and circulatory LH levels at environmentally relevant concentrations.

The bisphenol A (BPA)-disrupted reproductive functions have been demonstrated in male animals. In fish, it has been shown that environmentally relevant concentrations of BPA decrease sperm quality associated with inhibition of androgen biosynthesis. However, BPA effects on neuroendocrine regulation of reproduction to affect testicular functions are largely unknown. In the present study, reproductive functions of hypothalamus and pituitary were studied in mature male goldfish exposed to nominal 0.2, 2.0 and 20.0 µg/L BPA. At 90 d of exposure, sperm volume, velocity, and density and motility were decreased in goldfish exposed to 0.2, 2.0, and 20.0 µg/L BPA, respectively (p < 0.05). At 30 d of exposure, there were no significant changes in circulatory LH levels and mRNA transcripts of kiss1, Kiss2, gpr54, and gnrh3. At 90 d of exposure, circulatory LH levels showed trends toward increases in BPA exposed goldfish, which was significant in those exposed to 2.0 µg/L (P < 0.05). At this time, Kiss2, gpr54, and gnrh3 mRNA levels were increased in goldfish exposed to any concentrations of BPA (p < 0.05). This study shows that BPA-diminished sperm quality was accompanied by an increase in circulatory LH levels associated with increases in mRNA transcripts of upstream neuroendocrine regulators of reproduction in goldfish. Further, this is the first study to report circulatory levels of LH in fish exposed to BPA.

Roundup in the Reproduction of Crucian Carp (Carassius carassius): An In Vitro Effect on the Pituitary Gland and Ovary.

Roundup, the most popular herbicide in global agriculture, is regarded as an endocrine disruptor causing alterations of important hormones at the hypothalamic-pituitary-gonadal axis as well as impairment of gametogenesis. The whole pituitary glands of crucian carp (Carassius carassius) were incubated for 3 h in the medium containing Roundup (0-control, 1 and 10 ng/mL). The level of luteinizing hormone (LH), and mRNA transcript abundance of kisspeptin (kiss-1) and its receptor (gpr54), were determined. The isolated ovarian fragments were incubated for 24 h in the presence of Roundup and the following effects on reproductive parameters were determined: the final oocyte maturation and ovulation, structural changes in follicles, secretion of 17,20ß-progesterone (17,20ß-P) as well as mRNA transcript abundance of the luteinizing hormone receptor (lhr), estrogen receptors (erα, erß1, erß2), and zona radiata (chorion) proteins (zp2 and zp3). Roundup inhibited final oocyte maturation and decreased the percentage of ovulated eggs, and furthermore, caused structural changes in the ovarian follicular components. There were no significant changes in the measured hormone levels and analyzed genes mRNA transcript abundance. Summing up, obtained results indicate that Roundup may adversely affect oocyte maturation and the quality of eggs, suggesting that exposure to this herbicide can lead to reproductive disorders in fish.

Preliminary Results Concerning the Influence of Human Kisspeptin on LH Secretion in Prussian Carp (Carassius gibelio) Females at the Stage of Ovarian Recrudescence and Spawning Season.

The aim of the experiments was to evaluate the influence of human kisspeptin on LH secretion of Prussian carp (Carassius gibelio) females during ovarian recrudescence and spawning season. For the experiments, human kisspeptin KISS1 (0.1 mg kg(-1) of body weight--b.w.), GnRH analogue (Des Gly(10), D-Ala(6)) GnRH-A (20 µg kg(-1) b.w.) and dopamine antagonist (pimozide) (5 mg kg(-1) b.w.) were used alone or in combinations. At 3, 6, 12, 24 hours after injection(s) blood samples were collected from all fish. LH levels were measured in plasma with the use of the ELISA method. KISS1 did not show any significant effects on spontaneous LH secretion in both tested seasons. At 12 hours sampling time (both stages of gonad maturity) a combination of tested compounds (GnRH-A+KISS1) significantly increased LH release in comparison with the control. In the stage of gonad recrudescence KISS1 significantly increased LH secretion evoked by pimozide at 24 hours. A combination of three components: KISS1, GnRH-A, and pimozide significantly decreased LH secretion in comparison to LH secretion evoked by GnRH-A and pimozide during stage of gonad recrudescence. These results suggest that kisspeptin is involved in seasonal control of reproduction in Prussian carp. The possible interaction of kisspeptin and the dopaminergic system is also discussed.

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 17ß-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.

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 17ß-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 3ß-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.

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 effect of aromatase inhibitor, fadrozole, on sGnRHa stimulated LH secretion in goldfish (Carassius auratus) and common carp (Cyprinus carpio).

The aromatase inhibitor, fadrozole, was applied to common carp and goldfish in order to examine its ability to increase the spontaneous and sGnRHa stimulated LH secretion. First, trials in goldfish in 2003 showed fadrozole's moderate ability to potentiate sGnRHa-stimulated LH secretion. However, this ability was much weaker than that obtained with dopamine antagonist, pimozide. There was no ovulation in fadrozole-treated fish. Several experiments on goldfish and carp during the two consecutive years with the different treatment regimes and doses of fadrozole did not confirm the optimistic results obtained in 2003. This shows that fadrozole is unable to replace the antidopaminergic drugs being used in fisheries practice.

Differential effects of morphine and naltrexone on the in vitro LH secretion from male and female carp pituitary gland.

The in vitro effects of morphine (10(-10), 10(-8), 10(-6) or 10(-5) M) or/and naltrexone (10(-6) or 10(-8) M) on LH release from male and female carp (Cyprinus carpio L.) dispersed pituitary cells (obtained from fish at the time of late gonad recrudescence) were investigated. Morphine alone at the lowest tested concentration (10(-10) M) increased LH secretion from the cells of males. On the contrary, in female cell incubations the highest concentrations of morphine (10(-6) or 10(-5) M) significantly lowered LH levels. Naltrexone alone (at both tested concentrations) had no influence on LH secretion, neither in males nor in females. However in the incubations of female cells it antagonised the influence of morphine at 10(-10) or 10(-8) M. In male cell incubations naltrexone abolished the stimulatory action of morphine at 10(-10) M. The results suggest that in the in vitro culture of carp pituitary cells LH secretion is modulated by the opioids which affect the release of this gonadotropin through the typical opioid receptors and that the mu type of these receptors is involved in this process. The effects of opioid agonist and antagonist depend on the stage of gonadal maturity and the sex of fish i.e. the actual level of sex steroids.

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.

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.