Francisco Prat

Seasonal changes in plasma levels of gonadal steroids of sea bass, Dicentrarchus labrax L.

Levels of plasma testosterone (T) and 11-ketotestosterone (11-KT) in males and plasma 17 beta-estradiol (E2), 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P), and T in females were assayed by radioimmunoassay at monthly intervals throughout the sexual cycle of sea bass (Dicentrarchus labrax L.). 17 alpha,20 beta-DiOH-P was maintained at low levels (below 1 ng/ml) throughout the year, even during the spawning period (January-March). A bimodal seasonal pattern of plasma testosterone was observed. Plasma T and E2 levels became significantly increased in December (advanced gametogenesis period) and then showed further increases during January and February (first half of the spawning period) in parallel with the growth of the vitellogenic oocytes. Multiple spawnings of individual females were also observed during the spawning period affecting the relative fecundity of the eggs. A possible role of E2 on this behavior is discussed. In males, both plasma T and 11-KT initially increased in November and then showed further increasings during the rest of the period of gametogenesis (December) to reach their peak levels in the first half of the spawning period (end of January). These increased and sustained higher levels of plasma steroids coincided with the presence of spermiating males. A second peak of plasma testosterone appeared at the end of the postspawning period-beginning of the pregametogenesis period (May-June) both in males and females and their possible role with the preparation of the gonad for the next reproductive cycle is discussed.

Molecular Characterization of Putative Yolk Processing Enzymes and Their Expression During Oogenesis and Embryogenesis in Rainbow Trout (Oncorhynchus mykiss)

Abstract Vitellogenin is the major yolk protein precursor in fish, but little is known about its processing pathway in the oocyte, nor about mobilization of yolk proteins during embryogenesis. In this study we cloned three putative yolk processing enzymes; specifically, cathepsin B and L, and lipoprotein lipase (LPL), from the rainbow trout ovary and determined their patterns of gene expression, together with cathepsin D, during oogenesis and embryogenesis using reverse transcription-polymerase chain reaction. The approximate sizes of both cathepsin B and cathepsin L transcripts were estimated as 1.7–1.8 kilobases by Northern blot analysis. Cathepsin D mRNA and cathepsin L mRNA were expressed constitutively throughout vitellogenesis and embryogenesis, showing the highest levels of expression at around fertilization. Cathepsin B and LPL were expressed exclusively during oogenesis. Quantitatively, expression of cathepsin D mRNA was higher than cathepsin B, cathepsin L, and LPL mRNA throughout the period studied. The different patterns of expression for these genes during oogenesis and embryogenesis signify specific temporal roles in yolk protein processing.

The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarchus labrax L.)

Abstract Under simulated natural conditions female sea bass ( Dicentrarchus labrax ) in eastern Spain (latitude 40° N and longitude 0°) spawned over a 6–8-week period in February and March, with each female spawning on more than one occasion. Generally, male fish began spermiation up to 2 months before, and continued for at least 1 month after the spawning period of the female fish. Exposure of fish to 1 month of long days (LD 15 9 ) from either 2nd May (Group C), 3rd June (Group D) or 3rd July (Group E) in an otherwise constant short day (LD 9 15 ) photoperiod regime, speeded up the rates of maturation, thus increasing the proportions of oocytes entering exogenous vitellogenesis during October and November, and also brought forward the timings of ovulation and spawning. In contrast, constant long days from 2nd May (Group B) delayed maturation and spawning time by 2–3 months. Fish maintained under constant short days throughout the experiment (Group A) spawned up to 6 weeks in advance of the control fish, suggesting that endogenous timing mechanisms may operate in this species. Spawning occurred naturally in all the experimental and control fish without the necessity for induction with pituitary or hypothalamic hormones. For the control fish, egg quality (defined as the proportion of eggs spawned which were floating and viable), hatching rate and survival to first feeding (both expressed as percentages of the numbers of floating or good quality eggs) averaged 78.3±7.7% (mean±s.e), 84.8±3.3% and 54.6±20% respectively. With the exception of the eggs and fry from Group B, these measures of quality and survival appeared unaffected by the photoperiod treatments. The eggs and fry produced by the fish maintained under long days (Group B) towards the end of their spawning period (i.e. in May), showed much more variable egg quality and survivals through hatch and first-feeding, possibly because of the higher sea water temperatures which prevailed at the time of their delayed spawning. Fecundities expressed as numbers of eggs per kg of post-spawning fish weight ranged from 247–305 000/kg for Groups A, C, D and E and the controls, but were significantly reduced in Group B fish. Egg sizes, which ranged from 1.147–1.176 mm in diameter, were unaffected by the photoperiodic manipulations of spawning time. It is concluded that artificial control of daylength can improve the supply of eggs and fry for commercial on-growing without any adverse effects on the fecundity of the broodstock or the quality and survival of the eggs and fry.

Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development

The oocytes of many fish species accumulate high amounts of neutral lipids as a caloric reserve for embryonic and larval development. We propose that lipoprotein lipase (LPL, EC 3.1.1.34) plays an important role in supplying the oocytes with fatty acids and we have cloned its cDNA from the ovary of sea bass, and determined the patterns of LPL activity and LPL mRNA expression in the ovary. The cDNA obtained was 3051 bp long with an open reading frame encoding 518 amino acids. The amino acid sequence has a high similarity and shows similar structural features to LPL of other species. Northern blot analysis revealed LPL expression in adipose tissue and gonads only. LPL activity and LPL mRNA expression in the ovary was very high in fish with a gonadosomatic index (GSI) above 5, coinciding with the appearance of a high number of lipid droplets in the ooplasm. The LPL mRNA expression was localised to the follicle cells surrounding the oocyte. Our results suggest that LPL is likely to play an important role in the incorporation of neutral lipids into the oocytes, and that follicle cells, in addition to participating in steroidogenesis, also may be important in building up oocyte lipid reserves.

Molecular cloning of Senegalese sole (Solea senegalensis) follicle-stimulating hormone and luteinizing hormone subunits and expression pattern during spermatogenesis

Pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. However, in teleosts with testis of semi-cystic type and asynchronous spermatogenesis, as the flatfish Senegalese sole (Solea senegalensis), the physiological roles of FSH and LH are still not well understood. To gain insight into this mechanism, full-length complementary DNAs (cDNAs) encoding Senegalese sole FSH beta and LH beta subunits, and the common glycoprotein alpha subunit (CG alpha), were cloned and sequenced. The three cDNAs consisted of 550, 582 and 744 nucleotides encoding peptides of 120, 148 and 132 amino acids, respectively. Comparison of the deduced amino acid sequences of sole FSH beta, LH beta and CG alpha with those from other teleosts indicated that cysteine residues and potential N-linked glycosylation sites were fully conserved with respect to other percomorphs and salmonids. However, the primary structure of FSH beta and LH beta in pleuronectiforms appeared to be highly divergent. In situ hybridization of mature male pituitaries showed that fshb, lhb and cga mRNAs were localized in the proximal pars distalis and in the periphery of pars intermedia. Real-time quantitative reverse transcription-polymerase chain reaction indicated that the levels of all three transcripts in the pituitary of males increased during winter and spring, at the time when plasma levels of androgens raised and testicular germ cell development and spermatozoa production were stimulated. These results suggest that FSH and LH may regulate spermatogenesis in Senegalese sole similarly to that described for other teleosts with testis of cystic type and synchronous germ cell development.

Molecular characterisation of growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass

Members of the transforming growth factor-beta superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in primary follicle growth in mammals. To initiate investigations into their significance in teleost oogenesis, we set out to clone and characterise the cDNAs of gdf9 and bmp15 and analysed their patterns of gene expression during the ovarian reproductive cycle in the European sea bass (Dicentrachus labrax). Sea bass gdf9 and bmp15 cDNAs were 2200 and 2049 bp long, coding for 438 and 459 amino acids (aas), respectively, and were most similar to zebrafish gdf9 and bmp15 (64.4 and 56.1%, respectively). By Northern analysis, sea bass gdf9 and bmp15 mRNA transcripts were detected in the ovary only of the tissues analysed and their sizes were 2.2 and 2.1 kb, respectively. Dot-blot analysis revealed high levels of gdf9 and bmp15 expression in the ovary during primary oocyte growth and previtellogenesis (July to October), with a significant decline at the onset of vitellogenesis (November) and remaining low until the beginning of new oocyte growth (April/May). There was a highly significant positive correlation (r=0.939) between gdf9 and bmp15 gene expression in individual samples. The high levels of gdf9 and bmp15 mRNA transcripts in the ovary, especially during the previtellogenic growth period suggest an important role for these factors in early primary oocyte growth in the European sea bass.

Targeted gene expression profiling in European sea bass (Dicentrarchus labrax, L.) follicles from primary growth to late vitellogenesis

A real-time PCR-based gene expression survey was performed on isolated European sea bass follicles from primary growth to late vitellogenesis. Expression levels of 18 transcripts with demonstrated relevance during oogenesis, encoding gonadotropin, thyrotropin, estrogen, androgen, and vitellogenin receptors, steroidogenesis-related as well as growth and transcription factors were measured. Primary oocytes showed high mRNA levels of insulin-like growth factors 1 and 2, bone morphogenetic protein 4, estrogen receptor 2b, androgen receptor b, and SRY-box containing gene 17 together with low transcript amounts of gonadotropin receptors. Follicles at the lipid vesicles stage (i.e., the beginning of the secondary growth phase) showed elevated mRNA amounts of follicle stimulating hormone receptor (fshr) and anti-Mullerian hormone. Early-to-mid vitellogenic follicles showed high mRNA levels of fshr and cytochrome P450, family 19, subfamily A, polypeptide 1a while mid-to-late vitellogenic follicles expressed increasing transcript amounts of luteinizing hormone/choriogonadotropin receptor, steroidogenic acute regulatory protein, and estrogen receptors 1 and 2a. The molecular data presented here may serve as a solid base for future studies focused on unraveling the specific mechanisms orchestrating follicular development in teleost fish.

Nutritional and Photoperiodic Effects On Hormonal Cycles and Quality of Spawning in Sea Bass (Diceatrarchus Labrax L.)

Results are summarized concerning physiological responses of sea bass to different light and feeding regimes. Inappropriate broodstock management can cause significant alteration of hormonal rhythms with important consequences for the quality of eggs and larvae produced.

Estrogen-induced yolk precursors in European sea bass, Dicentrarchus labrax: Status and perspectives on multiplicity and functioning of vitellogenins.

The estrogen-inducible egg yolk precursor, vitellogenin, of the European sea bass (Dicentrarchus labrax) has received considerable scientific attention by virtue of its central importance in determination of oocyte growth and egg quality in this important aquaculture species. However, the multiplicity of vitellogenins in the sea bass has only recently been examined. Recent cloning and homology analyses have revealed that the sea bass possesses the three forms of vitellogenin, VtgAa, VtgAb and VtgC, reported to occur in some other highly evolved teleosts. Progress has been made in assessing the relative abundance and special structural features of the three Vtgs and their likely roles in oocyte maturation and embryonic nutrition. This report discusses these findings in the context of our prior knowledge of vitellogenesis in this species and of the latest advances in our understanding of the evolution and function of multiple Vtgs in acanthomorph fishes.

The role of lipoprotein lipase (LPL) in the incorporation of neutral lipids into the oocytes of the European sea bass (Dicentrarchus labrax L.) during gonadal development

We have cloned the cDNA encoding LPL and studied its mRNA expression and enzyme activity in the ovary of European sea bass in order to investigate a possible role of this enzyme in the incorporation of neutral lipids into the oocytes. The results suggest that LPL is likely to play an important role in this process.