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ABSTRACT: 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.
Molecular and Cellular Endocrinology 04/2008; 291(1-2):95-103. · 4.19 Impact Factor
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ABSTRACT: In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH/AMH), apart from inducing Müllerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5'-flanking region, and analysed its gene expression and those of alternatively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in juvenile testis, but still remarkably high in juvenile ovaries and adult testis, as well as in brain, pituitary, and heart of juvenile male sea bass. Apart from adult ovary, levels of alternatively-spliced sb-AMHexonII/-99 were marginal in comparison with sb-AMH. In contrast, the transcript variant sb-AMHexonVII/+5 was expressed to a similar extent as sb-AMH in all tissues examined. The results of this work have provided the basis for future studies concerning the regulation and function of AMH/AMH in this species.
Gene 03/2007; 388(1-2):148-58. · 2.34 Impact Factor
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ABSTRACT: The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) (P<0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) (P<0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.
Journal of Fish Biology 01/2006; 41(6):965 - 970. · 1.68 Impact Factor
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ABSTRACT: Under constant short photoperiod, the spawning time of 2-year-old sea bass Dicentrarchus labrax was advanced as compared to controls, whereas spawnings were delayed under constant long photoperiod. High plasma levels of 17β-oestradiol (E2/) and testosterone (T) in females were coincident with the appearance of vitellogenic oocytes in the ovary, while high levels of 11-ketotestosterone (11-KT) and T in males were coincident with the presence of spermiating males. Although plasma levels of E2 in females and 11-KT in males were low during the remainder of the cycle, levels of T were always >1 ng ml−1 in both sexes, suggesting that T could play an important role during the initial stages of gonadal development. The profiles of E2 and T in females and 11-KT and T in males exposed to constant short days were similar to those in the control group, but fish which were maintained under constant long photoperiods showed a bimodal pattern of these steroids. The results obtained from fish exposed to constant photoperiod regimes provide further evidence that an endogenous process could be operating to control the reproduction of sea bass.
Journal of Fish Biology 03/2005; 54(1):125 - 137. · 1.68 Impact Factor
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ABSTRACT: Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.
Molecular and Cellular Endocrinology 09/2004; 223(1-2):63-75. · 4.19 Impact Factor
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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.
Biology of Reproduction 01/2002; 65(6):1701-9. · 4.01 Impact Factor
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ABSTRACT: Partial cDNAs encoding two lipoprotein receptors were isolated and sequenced from the ovary in the rainbow trout (rt), Oncorhynchus mykiss. One of the cDNAs (rt-LPR) contained the 5 domains characteristic of receptors belonging to the low-density lipoprotein receptor superfamily. The second cDNA (rt-LP[OS]R) was similar to rt-LPR but lacked 105 base pairs encoding the O-linked sugar domain. The deduced amino acid sequences of the rt-LPR and rt-LP[OS]R had between 75% and 80% identity with very-low-density lipoprotein and vitellogenin receptors of other species. The rt-lipoprotein receptor mRNAs were approximately 3.5 kilobases in size. The rt-LPR was expressed in both the ovary and somatic tissues, whereas the rt-LP[OS]R was ovary-specific. Messenger RNA for the lipoprotein receptor(s) was expressed at high levels in both pre-vitellogenic (< 0.3 mm) and early vitellogenic (up to 1 mm) follicles. Using reverse transcription-polymerase chain reaction, expression of rt-LPR and rt-LP[OS]R mRNA was also detected in larger vitellogenic follicles (up to 2.5 mm in diameter) but not in follicles in late vitellogenesis or in ovulated eggs. The sequence, ovary specificity, and pattern of ovarian expression of the rt-LPR mRNA suggest that it encodes the receptor that mediates vitellogenin uptake into the ovary.
Biology of Reproduction 06/1998; 58(5):1146-53. · 4.01 Impact Factor
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ABSTRACT: Rainbow trout (Oncorhynchus mykiss) were subjected to unilateral ovariectomy (ULO) during early vitellogenesis to examine the endocrine responses mediating the recruitment and growth of oocytes in the secondary (vitellogenic) growth phase. ULO induced recruitment of a second population of primary oocytes into the vitellogenic growth phase that then grew at a faster rate than oocytes in the control fish. Seven days post-ULO, the concentration of plasma salmonid FSH (sFSH = GtH I) was significantly higher than in controls and was elevated for at least 54 days. Maximal concentrations of sFSH in ULO fish (Day 21 post-ULO) were twice (10 ng/ml) those in controls. The data show that sFSH plays a primary role in mediating vitellogenic development. After ULO, plasma concentrations of estradiol-17beta were significantly lower than in controls up until 21 days post-ULO. Thereafter, plasma concentrations of estradiol-17beta did not differ from those in controls. The changes in concentrations of plasma estradiol-17beta and sFSH in the ULO fish demonstrated that the secretion of sFSH is probably not controlled by negative feedback of estradiol-17beta alone; in fish, as in mammals, it is likely that intragonadal autocrine/paracrine factors, such as inhibin and activin, also participate in the regulation of sFSH secretion. Plasma concentrations of testosterone did not appear to differ between the control and ULO fish. The responses in the production of estradiol-17beta and testosterone indicate that the dynamics of sex steroid synthesis in ovarian follicles in ULO fish was different than in the ovaries of control fish.
Biology of Reproduction 12/1997; 57(5):1238-44. · 4.01 Impact Factor
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ABSTRACT: RIAs were developed for the two salmon gonadotropins (GTH I and GTH II) and used to measure plasma GTH throughout the life of the rainbow trout, Oncorhynchus mykiss. The RIA for GTH II was specific and sensitive (< 0.001% cross-reaction with GTH I, mean sensitivity = 0.26 +/- 0.02 ng/ml). The RIA for GTH I was less specific and less sensitive than the GTH II RIA (9.7% cross-reaction with GTH II, mean sensitivity = 2.34 +/- 0.23 ng/ml). In both males and females, the levels of GTH II remained undetectable (< 0.3 ng/ml) throughout most of the reproductive cycle, until shortly preceding spermiation/ovulation, when they began to rise. Concentrations of plasma GTH II were maximal at spermiation/ovulation. In both sexes, plasma profiles of GTH I differed from those of GTH II. The plasma GTH I concentration in females was elevated during early vitellogenesis. It then fell to a basal level shortly before ovulation and finally was elevated again at ovulation. In males, increases in plasma GTH I were seen a year before spermiation and again later, during the final stages of testicular growth. These results support the contention that GTH I mediates gonadal growth, whereas GTH II regulates the final stages of maturation and ovulation/spermiation. In rainbow trout, plasma profiles of GTH I and GTH II mimic the cycles of plasma FSH and LH, respectively, in the ovulatory cycle of higher vertebrates.
Biology of Reproduction 07/1996; 54(6):1375-82. · 4.01 Impact Factor
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ABSTRACT: 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.
Netherlands Journal of Zoology 12/1993; 45(1-2):204-209.
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ABSTRACT: 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.
General and Comparative Endocrinology 07/1990; 78(3):361-73. · 3.27 Impact Factor
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ABSTRACT: Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERα, and two ERβ subtypes, called ERβ1 and ERβ2 (or ERβ and ERγ in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERα cDNA) and ovary (partial sb-ERβ1 and sb-ERβ2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERα, ERβ1 and ERβ2. Deduced amino acid numbers for sb-ERα, sb-ERβ1 and sb-ERβ2 were 639, 517 and 608, respectively, representing in the case of sb-ERβ1 and sb-ERβ2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERα with eelpout ERα (88.7%), for sb-ERβ1 with Atlantic croaker ERγ (85.8%), and for sb-ERβ2 with Atlantic croaker ERβ (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERα was predominantly expressed in liver and pituitary, while sb-ERβ1 and sb-ERβ2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERα expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERβ1 and sb-ERβ2 highest expression levels were observed in gonads at 250 dph. For sb-ERβ2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.
Molecular and Cellular Endocrinology.
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ABSTRACT: To determine the influence of time of day on the ovarian activity of LHRHa-treated sea bass females, two groups were injected with 10 + 10 μg/kg body weight 24 h apart at either 10.00 or 22.00 h. The response was evaluated by the course of oocyte maturation, ovulation and plasma levels of 17 β-estradiol (17 βE2), testosterone (T) and 17α20β-dihydroxyprogesterone (17-20-P) compared to saline-injected control groups. Both LHRHa-treated groups exhibited a marked increase in the proportion of germinal vesicle breakdown in oocytes at 16 (group 10.00) and 24 h (group 22.00) after the first LHRHa injection. Ovulation began at 32 h, in 80% of group 10.00 and 20% of group 22.00, reaching 100% in both groups at 48 h. Control groups did not ovulate throughout the experiment. Treated females showed a T peak at 8 and a 17 βE2 peak at 16 h after the first LHRHa injection, the 17 βE2 plasma levels being higher for the 22.00 than for the 10.00 group. Data obtained support the hypothesis that 17 βE2 acts as a negative factor on final oocyte maturation and ovulation, and that there is a daily change in sensitivity either for the pituitary to LHRHa or for the ovary to gonadotropin. Plasma levels of 17α20β-P were not detectable, confirming that this steroid does not act as maturation-inducing steroid (MIS) in the ovulating sea bass. Further research is necessary to establish the real advantage of giving the LHRHa injections in the morning.
Aquaculture.
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ABSTRACT: Members of the transforming growth factor-β 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.
Molecular and Cellular Endocrinology.
