Male Seabream Research Articles

17\u03b1-ethynylestradiol prevents the natural male-to-female sex change in gilthead seabream (Sparus aurata L.)

Exposure to 17α-ethynylestradiol (EE2, 5 μg/g food) impairs some reproductive events in the protandrous gilthead seabream and a short recovery period does not allow full recovery. In this study, spermiating seabream males in the second reproductive cycle (RC) were fed a diet containing 5 or 2.5 μg EE2/g food for 28 days and then a commercial diet without EE2 for the remaining RC. Individuals were sampled at the end of the EE2 treatment and then at the end of the RC and at the beginning of the third RC, 146 and 333 days after the cessation of treatment, respectively. Increased hepatic transcript levels of the gene coding for vitellogenin (vtg) and plasma levels of Vtg indicated both concentrations of EE2 caused endocrine disruption. Modifications in the histological organization of the testis, germ cell proliferation, plasma levels of the sex steroids and pituitary expression levels of the genes coding for the gonadotropin β-subunits, fshβ and lhβ were detected. The plasma levels of Vtg and most of the reproductive parameters were restored 146 days after treatments. However, although 50% of the control fish underwent sex reversal as expected at the third RC, male-to female sex change was prevented by both EE2 concentrations.

Endocrine disrupter chemicals affect the humoral antimicrobial activities of gilthead seabream males even upon the cease of the exposure

17α-ethynilestradiol (EE2) and tamoxifen (Tmx) are pollutants world-wide distributed in aquatic environments. Gilthead seabream, Sparus aurata L., is highlighted as a species model of intensively culture in anthropogenic disturbed environments. The effects of these pollutants on gilthead seabream reproduction and some immune responses have been described but, the humoral innate antimicrobial activities have never received attention. In this work we analysed the latest in the plasma of gilthead seabream males of different ages and reproductive stages treated with 0, 2.5, 5 or 50 μg EE2 or 100 μg Tmx g−1 food during different times of exposure and of reverting to commercial diet (recovery). The peroxidase and protease activities decreased as the spermatogenesis of the first reproductive cycle (RC) proceeded in control fish. However, only protease and antiprotease activities showed different level at different stages of the second RC in control fish, but showed scarce disruption in fish treated with EE2 or Tmx. Peroxidase and bactericide activities are more sensitive to EE2, than to Tmx. The effects induced by EE2 varied depending on the activity analyzed, the dose and the time of exposure and the reproductive stage and the age of the specimens.

Genetic relationships and hybridization among three western Atlantic sparid species: sheepshead (Archosargus probatocephalus), sea bream (A. rhomboidalis) and pinfish (Lagodon rhomboides)

Three species of sparids in the western Atlantic, sheepshead (Archosargus probatocephalus), sea bream (A. rhomboidalis), and pinfish (Lagodon rhomboides), share overlapping habitats, spawning seasons, and spawning grounds, providing opportunities for interaction among these species. Three regions of mitochondrial DNA and three nuclear DNA intron sequences were used to construct the genetic relationships among these species. The results showed that these species are closely related, suggesting the presence of soft polytomy with sheepshead and western Atlantic sea bream as sister species. However, western Atlantic sea bream and pinfish are equally divergent from sheepshead. We used a suite of 18 microsatellite markers to verify the occurrence of hybridization, identify the parental types, and evaluate the filial-generation status of 36 individuals morphologically identified as hybrids from the Indian River Lagoon system, in Florida. The 36 putative hybrids were analyzed with a reference group of 172 western Atlantic sea bream, 232 pinfish, and 157 sheepsheads and were all genetically determined to be F1 of sheepshead and western Atlantic sea bream with very little indication or no introgressive hybridization among the 172 reference specimens of western Atlantic sea bream. Hybridization was asymmetric, with western Atlantic sea bream males crossing with sheepshead females. Hybrids were first observed in the Indian River Lagoon in 2005, after the western Atlantic sea bream had become common there, in the 1990s. Their occurrence could be associated with unique features of the Indian River Lagoon that bring the two species together or with recent anthropogenic changes in this system. Further study is needed to determine the causes and long-term effects of the recurrent production of F1 hybrids and the degree of their sterility in the Indian River Lagoon.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

Tamoxifen disrupts the reproductive process in gilthead seabream males and modulates the effects promoted by 17α-ethynylestradiol

17α-Ethynylestradiol (EE2), which is used in oral contraceptives and hormone replacement therapy, is a well documented estrogenic endocrine disruptor and an aquatic contaminant. In the present study, adult male specimens of the marine hermaphrodite teleost gilthead (Sparus aurata L.) were fed a diet containing tamoxifen (Tmx), an estrogen receptor ligand used in cancer therapy, alone or combined with EE2, for 25days and then fed a commercial diet for a further 25days (recovery period). The effects of short (5days) and long (25days) treatments on several reproductive and gonad immune parameters and the reversibility of the disruptive effects after the recovery period were examined. Our data showed that Tmx acted as an estrogenic endocrine disruptor as revealed by the increase in the hepatic transcription of the vitellogenin gene in males, the serum levels of 17β-estradiol and the gonad expression levels of the estrogen receptor α and G protein-coupled estrogen receptor genes, and the recruitment of leukocytes into the gonad, a well known estrogenic-dependent process in gilthead seabream males. On the other hand, Tmx also increased sperm concentration and motility as well as the serum levels of androgens and the expression levels of genes that codify for androgenic enzymes, while decreasing the expression levels of the gene that code for gonadal aromatase. When applied simultaneously, Tmx and EE2 could act in synergy or counteract, each other, depending on the parameter measured. The disruptive effect of EE2 and/or Tmx was not reversible after a 25day recovery period.

Dietary intake of 17α-ethinylestradiol promotes leukocytes infiltration in the gonad of the hermaphrodite gilthead seabream

A wide variety of chemicals discharged from industrial and municipal sources have been reported to disrupt the endocrine system of animals, which may be exposed via the food chain and contaminated water. 17α-Ethinylestradiol (EE2), a drug used in oral contraceptives and hormone replacement therapy, has a widespread presence in the aquatic environment. Current knowledge on the sensitivity of marine fish to estrogenic environmental chemicals is limited. We report here the effects of dietary intake of EE2 on gilthead seabream, a marine hermaphrodite teleost, focusing on the immune events that take place in the gonad. When seabream males were fed with 5, 50, 125 and 200μg EE2/g food for 7, 14, 21 and 28days an infiltration of acidophilic granulocytes and B lymphocytes occurred in the testis as the same time that spermatogenesis is disrupted. Moreover, the dietary intake of EE2 promoted a dose-dependent up-regulation of the expression of genes coding for cytokines, chemokines and adhesion molecules correlated with a leukocyte infiltration.

Body lipid and fatty acid composition in male gilthead seabream broodstock at different stages of the reproductive cycle: effects of a diet lacking n-3 and n-6 HUFA

Total lipid (TL), lipid classes and fatty acid composition of neutral (NL) and polar (PL) lipids were studied in the gonads, liver and muscle of gilthead seabream males (Sparus aurata) fed a control diet (diet C) or an n-3 and n-6 highly unsaturated fatty acids (HUFA)-deficient diet (diet D), at different stages of the reproductive cycle. Between pre-spermatogenesis (November) and spermatogenesis (March), the lipid content was high and particularly rich in cholesterol, phosphatidylcholine and phosphatidylethanolamine in gonads from both dietary groups. At post-spermatogenesis (June), TL and especially PL dramatically decreased in the gonads from both groups. However, at this period diet C fish gonads were richer in triacylglycerides (TAG) than those from diet D fish. The liver lipid contents and particularly TAG were over 200% lower in June than in March for both groups. Nevertheless, the most noteworthy depletion of lipids during this period was achieved by the n-3 HUFA in diet D fish. Conversely, arachidonic acid (20:4n-6) did not decrease in NL or PL from gonads and liver in groups C and D. Muscle lipids from diet C fish were relatively insensitive to seasonal influences. However, in June, the muscle TAG content was significantly reduced in diet D fish.

Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post-thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing-resistant (or intact) and eliminating non-viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo-methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen-thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post-thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo-injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.

Lipid and fatty acid content in wild white seabream ( Diplodus sargus) broodstock at different stages of the reproductive cycle

The lipid and fatty acid content of the gonads, liver and muscle of wild white seabream males and females was studied at different stages of the reproductive cycle. Samples were taken from mature white seabream at pre-spawning (November), mid-spawning (March) and post-spawning (June) stages. The results showed that lipid accumulates in gonads and muscle from November to March. The gonadosomatic index (GSI) was also increased during this period. Male gonads showed a greater increase in polar lipid (PL) than neutral (NL), while female gonads displayed the reverse. The increase in both neutral and polar lipid was higher in the muscle of males than in females. In the same period, male livers showed no changes either in lipid content or the hepatosomatic index (HSI), while female livers registered an increase in both lipid content and HSI. Between March and June, in both males and females, total, neutral and polar lipid decreased sharply in the gonads and muscle. Muscular lipid content reduction was more pronounced in males than females. On the other hand, the lipid content of the liver in males and females remained relatively constant. In general terms, the amounts of major fatty acids (16:0, 18:1n-9, 20:5n-3 and 22:6n-3) in gonadal and muscular polar and neutral lipid in both males and females increased from November to March and declined thereafter. Variations of the liver fatty acid content were less extreme. In the period from mid-spawning to post-spawning, the presence of 20:4n-6 in polar and neutral lipid increased to a notable extent in all organs studied.

17Beta-Estradiol Triggers Postspawning in Spermatogenically Active Gilthead Seabream (Sparus aurata L.) Males1

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6-14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.