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Effects of reproductive stage and 11-ketotestosterone on LPL mRNA levels in the ovary of the shortfinned eel

2010; Elsevier BV; Volume: 51; Issue: 11 Linguagem: Inglês

10.1194/jlr.m009027

1539-7262

Sean L. Divers, H. James McQuillan, Hajime Matsubara, Takashi Todo, P. Mark Lokman,

Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities

To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT. To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT. It is well established in teleosts that lipids form the major component of the body nutrient pool in terms of energy storage (1Watanabe T. Lipid nutrition in fish.Comp. Biochem. Physiol. 1982; 73: 3-15Google Scholar, 2Sheridan M.A. Kao Y.H. Regulation of metamorphosis associated changes in the lipid metabolism of selected vertebrates.Am. Zool. 1998; 38: 350-368Crossref Scopus (104) Google Scholar). The main lipid storage sites in fish include liver, muscle, and mesenteric fat (2Sheridan M.A. Kao Y.H. Regulation of metamorphosis associated changes in the lipid metabolism of selected vertebrates.Am. Zool. 1998; 38: 350-368Crossref Scopus (104) Google Scholar), but the relative importance of each tissue depot varies among fish species (3Navarro I. Gutierrez J. Fasting and Starvation.in: Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fishes. Elsevier Science, Amsterdam1995: 393-434Google Scholar). 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Garfinkel A.S. Schotz M.C. Lipolytic enzymes and plasma lipoprotein metabolism.Annu. Rev. Biochem. 1980; 49: 667-693Crossref PubMed Scopus (570) Google Scholar)). As the catabolism of FFA provides the main source of energy for many species (11Tocher D.R. Metabolism and functions of lipids and fatty acids in teleost fish.Rev. Fish. Sci. 2003; 11: 107-184Crossref Scopus (1709) Google Scholar), LPL is considered a key enzyme in whole body lipid metabolism and balance, and the extra-hepatic rate-limiting enzyme in the hydrolysis of circulating TAG (12Blanchette-Mackie E.J. Masuno H. Dwyer N.K. Olivecrona T. Scow R.O. Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study.Am. J. Physiol. 1989; 256: E818-E828PubMed Google Scholar, 13Enerback S. Gimble J.M. Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level.Biochim. Biophys. Acta. 1993; 1169: 107-125Crossref PubMed Scopus (176) Google Scholar, 14Albalat A. Sanchez-Gurmaches J. Gutierrez J. Navarro I. Regulation of lipoprotein lipase activity in rainbow trout (Oncorhynchus mykiss) tissues.Gen. Comp. Endocrinol. 2006; 146: 226-235Crossref PubMed Scopus (67) Google Scholar).Aside from their importance in generating energy, the lipid composition of eggs is a key determinant of egg quality in marine fish (reviewed in Ref. 15Rainuzzo J.R. Reitan K.I. Olsen Y. The significance of lipids at early stages of marine fish: a review.Aquaculture. 1997; 155: 103-115Crossref Scopus (388) Google Scholar); however, there is almost no information about when and how neutral lipids are incorporated into the oocyte or the role that LPL plays in this event. Likewise, the fate and, in particular, the movement of neutral lipid from storage depots to the ovary throughout oogenesis is largely unknown, despite the obvious relevance of these issues for aquaculture operations. Freshwater eels are highly suitable experimental models for the study of lipid movement and uptake as they cease feeding at the onset of puberty. Thus, eels rely totally on endogenous TAG stores to both fuel a long-distance spawning migration and complete gametogenesis.We aimed (i) to determine the potential involvement of LPL in lipid uptake into the eel ovary throughout (induced) oogenesis, and (ii) to investigate the effects of treatment with 11-ketotestosterone (11-KT). The latter aim was proposed in view of the documented effects of 11-KT on lipid uptake in vivo (16Rohr D.H. Lokman P.M. Davie P.S. Young G. 11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis.Comp. Biochem. Physiol. 2001; 130A: 701-714Crossref Scopus (96) Google Scholar) and in vitro (17Lokman P.M. George K.A. Divers S.L. Algie M. Young G. 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro.Reproduction. 2007; 133: 955-967Crossref PubMed Scopus (124) Google Scholar, 18Endo T. Todo T. Lokman P.M. Ijiri S. Adachi S. Yamauchi K. In vitro induction of oil droplet accumulation into previtellogenic oocytes of Japanese eel, Anguilla japonica.Cybium. 2008; 32: 239-240Google Scholar) and on the demonstrated ability of 11-KT to increase eel oocyte diameters in vitro (17Lokman P.M. George K.A. Divers S.L. Algie M. Young G. 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro.Reproduction. 2007; 133: 955-967Crossref PubMed Scopus (124) Google Scholar).MATERIALS AND METHODSAnimals and experimental designExperiments were conducted within the guidelines of the University of Otago Animal Ethics Committee (protocols 32/05 and 51/05).Experiment 1: Lipid content, LPL mRNA levels, and LPL enzyme activity in the ovary and liver of wild shortfinned eelsEight previtellogenic (PV; "yellow") and eight early vitellogenic (EV; "silver") shortfinned eels (A. australis) were captured during early summer and early autumn, respectively, to obtain baseline data on lipid physiology. Eels were caught by commercial fyke nets set overnight in Lake Ellesmere, Christchurch, New Zealand and euthanized in 0.3 g/l benzocaine prior to sampling. Body length and body, liver, and gonad weights were determined for purposes of normalization and calculation of the gonadosomatic index (GSI; gonad weight as percentage of total body weight). After removal of the ovary, a small amount of tissue was fixed in phosphate-buffered 4% paraformaldehyde for oocyte staging. Ovarian tissue was snap-frozen in liquid nitrogen and stored at −80°C until determination of total lipid content, LPL activity, and quantitative PCR (qPCR).Experiment 2: Lipid content, LPL mRNA levels, and LPL enzyme activity in the ovary and liver of artificially maturing shortfinned eelsAs eels do not undergo vitellogenesis in captivity and mature eels cannot be sourced from the wild (19Dufour S. Le Belle N. Baloche S. Fontaine Y-A. Positive feedback-control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels, Anguilla anguilla.Fish Physiol. Biochem. 1989; 7: 157-162Crossref PubMed Scopus (39) Google Scholar), an induced-maturation trial was carried out, which allowed for the sampling of eels in different stages of reproductive development. Twenty-three wild-caught EV eels were purchased from a commercial eel processor (Gould Aquafarms, Christchurch, New Zealand) and housed in 1,000 l recirculating tanks. Water temperature was maintained at 20°C and salinity at 30 ppt. Fish were injected intramuscularly once every two weeks with 10 mg·kg-1 salmon pituitary homogenate (SPH) (20Lokman P.M. Young G. Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla dieffenbachiiA. australis).N. Z. J. Mar. Freshw. Res. 2000; 34: 135-145Crossref Scopus (60) Google Scholar), consisting of acetone-dried Chinook salmon pituitary homogenized in eel Ringer (for Ringer composition, see Ref. 21Miura T. Yamauchi K. Takahashi H. Nagahama Y. Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel, Anguilla japonica.Proc. Natl. Acad. Sci. USA. 1991; 88: 5774-5778Crossref PubMed Scopus (433) Google Scholar). Control fish were injected with an equivalent volume of eel Ringer solvent only. Three fish were induced to undergo final oocyte maturation and ovulation using the method outlined in Ref. 20Lokman P.M. Young G. Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla dieffenbachiiA. australis).N. Z. J. Mar. Freshw. Res. 2000; 34: 135-145Crossref Scopus (60) Google Scholar. SPH-treated eels were sampled at various stages of oocyte development after euthanasia in 0.3 g·l−1 benzocaine; Ringer-injected controls were sampled at the same time. Attempts were made to sample ovarian tissue from three fish at each developmental stage [e.g., EV; mid-vitellogenesis (MV); late vitellogenesis (LV); migratory nucleus (MN); and postovulation (P-OV)], but retrospective oocyte staging indicated that this was not achievable in all instances. Likewise, insufficient animals were available for sampling at week 10, and only two control fish were available for sampling when SPH-treated fish were ovulating. After removal of the ovary, tissue was sampled as described above (Experiment 1).Experiment 3: Effects of 11-KT on ovarian and liver LPL mRNA levels and lipid content in vivoFourteen PV shortfinned eels were divided between two treatment groups and implanted intraperitoneally with a pellet (95% cholesterol, 5% cellulose) containing placebo or 2.5 mg 11-KT, as described previously (22Lokman P.M. Kazeto Y. Ozaki Y. Ijiri S. Tosaka R. Divers S.L. Matsubara H. Moore L.G. Adachi S. Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis.Reproduction. 2010; 139: 71-83Crossref PubMed Scopus (22) Google Scholar). Fish were maintained for two weeks in fresh water at 16–17°C before terminal anesthesia in 0.3 g·l−1 benzocaine. Liver and ovarian tissues were collected and frozen for quantification of LPL mRNA levels by qPCR.Experiment 4: Effects of 11-KT on ovarian LPL mRNA levels in vitroIn vitro trials were run on ovarian tissue from PV shortfinned female eels, in the presence or absence of 11-KT. Five females were used for both experiments 4.1 and 4.2, and ovarian cultures were maintained for 18 days, whereas experiment 4.3 utilized ovaries from six fish maintained for 3 days, all at 16°C. Ovarian fragments were retrieved and incubated in a supplemented L-15 medium in vitro in a dose response design (0–1,000 nM 11-KT) (experiment 4.1) or in the presence or absence of 100 nM 11-KT as described previously (17Lokman P.M. George K.A. Divers S.L. Algie M. Young G. 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro.Reproduction. 2007; 133: 955-967Crossref PubMed Scopus (124) Google Scholar) (experiments 4.2 and 4.3). At the end of incubation, tissues were recovered and frozen for measurement of LPL mRNA levels by qPCR.Histological analysisFixed tissue was dehydrated, embedded in paraffin, and sectioned at 5–6 μm according to standard procedures. Sections for oocyte staging were stained with either hematoxylin and eosin or Mallory trichrome. Oocytes whose nuclei were visible in the section were staged as previously described (20Lokman P.M. Young G. Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla dieffenbachiiA. australis).N. Z. J. Mar. Freshw. Res. 2000; 34: 135-145Crossref Scopus (60) Google Scholar, 23Guraya S.S. Kaur R. Saxena P.K. Morphology of ovarian changes during the reproductive cycle of the fish, Mystus tengara (Ham.).Acta Anat. (Basel). 1975; 91: 222-260Crossref PubMed Google Scholar, 24Kazeto Y. Ijiri S. Matsubara H. Adachi S. Yamauchi K. Cloning of 17β-hydroxysteroid dehydrogenase-I cDNAs from Japanese eel ovary.Biochem. Biophys. Res. Commun. 2000; 279: 451-456Crossref PubMed Scopus (38) Google Scholar). The most developmentally advanced oocytes within the ovary determined the oocyte stage assigned to individual fish.Determination of total lipid contentLipid content was quantified using an adaptation of the method used by Folch et al (25Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Ovarian or liver tissue (100–150 mg) was macerated using razor blades and transferred to a test tube containing 4 ml CHCl3:MeOH (2:1). After mixing by vortex for 60 s, 1 ml 0.8% NaCl was added, and mixing was repeated for 60 s. Following centrifugation at 4,000 g for 4 min, the lower CHCl3 phase was transferred to a preweighed 4 ml glass tube. The CHCl3 was evaporated, and after desiccation for 24 h, the tubes were weighed to determine the total amount of lipid present.LPL activity assayLPL activity was measured in ovarian tissue collected from eels in experiments 1 and 2. Ovarian homogenates (for details, see Ref. 26Ibáñez A.J. Peinado-Onsurbe J. Sánchez E. Cerdá-Reverter J.M. Prat F. Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development.Comp. Biochem. Physiol. 2008; 150A: 347-354Crossref Scopus (54) Google Scholar) were centrifuged at 12,000 g for 2 min at 4°C, and then the supernatant was removed and placed on ice for immediate use. Two aliquots of homogenate from each eel were assayed: one aliquot contained apoCII (from VLDL of human plasma; Merck) in apoCII buffer (10 mM Tris-HCl, pH 8.5) to stimulate LPL activity (27Lindberg A. Olivecrona G. Lipoprotein lipase from rainbow trout differs in several respects from the enzyme in mammals.Gene. 2002; 292: 213-223Crossref PubMed Scopus (60) Google Scholar); the other aliquot contained the equivalent volume of only apoCII buffer to estimate LPL-independent lipase activity. The assay mixture consisted of 0.016 mM [carboxyl-14C] triolein (102 mCi/mmol; Perkin-Elmer, Wellesley, MA); 50 mM MgCl2; 25 mM PIPES (pH 7.5); 0.05% BSA (FA-free); 3 μg apoCII or apoCII buffer only; and 7 μl of ovarian homogenate in a total volume of 67 μl. After incubation at 25°C on a shaking platform for 2 h, the reaction was stopped, and the oleate was extracted. After centrifugation (1,000 rpm for 15 min), the supernatant was removed and measured for radioactivity by scintillation counting (Trilux Scintillation and Luminescence Counter, Wallac). LPL activity was calculated as the difference between total and LPL-independent oleate release, and expressed in mU. As some tissue from wild PV and EV eels was used up to develop and validate the assay, quantitative data from these groups could be gathered only from four of eight fish in each group.Cloning of partial A. australis LPL cDNATotal RNA was extracted from an ovary of an EV eel according to the Trizol® protocol (Invitrogen). Complementary DNA was generated from 1 μg total RNA using oligo-dT18 and Superscript III (Invitrogen) reagents. A 474 bp fragment of eel LPL cDNA was amplified by PCR using degenerate primers designed around a conserved region of published teleost LPL sequences and an A. japonica LPL sequence (T. Todo, unpublished observations). The sequence for the forward primer was 5′-TNATYCATGGRTGGAC -3′. The sequence for the reverse primer was 5′-CCRTTRGGGTAGATRTC-3′. The PCR protocol consisted of 2 min at 94°C, followed by 30 cycles of 94°C for 15 s, 47°C for 20 s, 72°C for 40 s, and a final extension of 3 min at 72°C, utilizing Bioline reagents. The amplified PCR product was extracted from an agarose gel with the QIAEX II Agarose Gel Extraction Kit (QIAGEN, Hilden, Germany) and ligated into the pGEM®-T Easy Vector (Promega, Madison, WI). After transfection and amplification in competent E. coli (strain XL-1 Blue), plasmid was isolated using the Plasmid Mini Kit (QIAGEN, Hilden, Germany) and sequenced (Allan Wilson Centre, Massey University, Palmerston North, New Zealand). The identity of a 441 bp cDNA (474–33 bp from degenerate primer sequences) was confirmed (BLAST alignment, NCBI), and nested primers (forward: 5′-CACCTCCGCTGCTACACCAAGCTTGT-3′; reverse: 5′-GTTGGGGTAGATGTCCACGTAGCCCA-3′) were subsequently designed for phylogenetic and sequence analysis. An amplicon (339 bp) was obtained by PCR using the cDNA, reaction volume, and reagents described previously. The PCR protocol consisted of 2 min at 94°C, followed by 30 cycles of 94°C for 15 s, 68°C for 20 s, and 72°C for 35 s, followed by a final extension of 3 min at 72°C. The nested PCR product was sequenced as described above and aligned with other lipases to ascertain homology using the neighbor joining method (DNASIS v3.5).Real-time PCR for quantification of LPL mRNA levelsTranscript levels were measured by real-time quantitative PCR (Stratagene Mx3000P, Agilent Technologies, CA). The reaction mixture contained 50 ng reverse-transcribed RNA, 50 nM forward primer (5′-AGCTTCACCTTCTGGGATACAG-3′), 150 nM reverse primer (5′-TGTTTGGTGTTCTAGTTGTCCT-3′), 10 μl Platinum SYBR Green Supermix (Invitrogen, Auckland, New Zealand), and distilled water to a final volume of 20 μl. All reactions were carried out in duplicate (LPL) or triplicate [elongation factor-1α (ELF-I)]. The PCR protocol consisted of 2 min at 50°C, 2 min at 95°C, followed by 30 cycles of 95°C for 15 s, 61°C for 30 s, and 72°C for 30 s, followed by 1 min at 95°C, 30 s at 55°C, and 95°C for 30 s After estimation of the copy number using a standard curve with known amounts of linearized pGEM T-Easy-LPL construct, LPL transcript levels were expressed as a whole-organ copy number or normalized over mg wet tissue weight, over μg total RNA, or over the housekeeping gene ELF-I (17Lokman P.M. George K.A. Divers S.L. Algie M. Young G. 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro.Reproduction. 2007; 133: 955-967Crossref PubMed Scopus (124) Google Scholar, 22Lokman P.M. Kazeto Y. Ozaki Y. Ijiri S. Tosaka R. Divers S.L. Matsubara H. Moore L.G. Adachi S. Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis.Reproduction. 2010; 139: 71-83Crossref PubMed Scopus (22) Google Scholar) as appropriate (see detailed outline below).As the full-length cDNA sequences for LPL and ELF-I are not available for the shortfinned eel, primers could not be optimally designed to cover intron/exon boundaries to control for genomic contamination. Therefore, possible contamination with genomic DNA in each sample was estimated by qPCR assay of RNA extracts (nonreverse-transcribed). Genomic contamination was less than 0.1% for LPL and less than 0.003% for ELF-I.Data transformation and statistical analysisExperiments 1 and 2.The morphometric changes in female eels undergoing gonadal maturation are not isometric. Eel total length is known not to change during maturation, but body weight, width, and depth do change, prompting the use of an allometric eel model (AEM) to correct the various lipid and gene transcript abundance analyses for body size. First, the relationship between body weight and total body length [condition index (CI)] was established using an ordinary least squares regression (28Packard G.C. Boardman T.J. The misuse of ratios, indices, and percentages in ecophysiological research.Physiol. Zool. 1988; 61: 1-9Crossref Google Scholar, 29Cone R.S. The need to reconsider the use of condition indices in fishery science.Trans. Am. Fish. Soc. 1989; 118: 510-514Crossref Google Scholar) based on measurements of female shortfinned eels collected from Lake Ellesmere by members of our laboratory over the last decade or so. The regression equation, fitted only to fish (n = 99) whose total length fell within the range of 565–982 mm of the fish used in the current study, was determined on the basis of Le Cren (30Le Cren E.D. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch Perca fluviatilis.J. Anim. Ecol. 1951; 20: 201-219Crossref Google Scholar) and Cone (29Cone R.S. The need to reconsider the use of condition indices in fishery science.Trans. Am. Fish. Soc. 1989; 118: 510-514Crossref Google Scholar) to model weight (W) in mm as a function of length (L) in mm, yielding condition index (CI): CI=(W/Lb)∗10nwhere b equals the slope of the least squares regression, and n is chosen so that CI is a mixed number instead of a very low decimal figure (31Anderson R.O. Gutreuter S.J. Length, weight, and associated structural indices.in: Nielsen L.A. Johnson D.L. Fisheries Techniques. American Fisheries Society, Bethseda, MD1983: 283-300Google Scholar). For shortfinned eel, these parameters were estimated as b = 2.91 and n = 5.The slope from the regression equation was used to generate a standardized, allometric eel length: AEM=L2.91where total length L is measured in meters. This model provides the best possible standardization for body size, given that it is sex-, size-, and location-specific to the fish used in this study. Standardization was achieved by dividing whole-animal or organ lipid or transcript data by the AEM. Use of ELF-I as a normalizer was not possible for ovarian data from experiments 1 and 2 (ANOVA indicated that mRNA levels differed significantly between developmental stages); therefore, total RNA was used as a normalizer instead. Given our interest in the total capability to accrue lipids, most of our data are not normalized over ELF-I, but rather, presented as a whole-organ mRNA copy number.Data were evaluated for normality using P-P plots and Shapiro-Wilk analysis and for homogeneity of variance using Levene's test. Wild eel data were analyzed using Student's 2-tailed t-test. Data from experiment 2 were analyzed using one-way ANOVA (for this purpose, all controls were grouped together), followed by Hochberg's posthoc tests to account for the largely different sample sizes among groups and by Games-Howell test to account for lack of homogeneity of variance.Experiments 3 and 4.Eels used in experiment 3 had comparable body weights, obviating the need to apply AEM corrections. Data were tested for normality and homogeneity of variance (as above), and following normalization over the ELF-I copy number, analyzed by ANOVA to compare treatment means. Likewise, in vitro data from experiment 4 were analyzed following normalization over ELF-I by ANOVA, using treatment (fixed variable) and fish (random variable) in the model.RESULTSLPL cDNA sequence analysisA partial cDNA, obtained by nested PCR, of 339 bp (accession no. HM775157) could be translated into a deduced amino acid sequence of 113 residues that corresponded to residues 143–255 of zebrafish LPL (Danio rerio; NP_571202). The deduced amino acid sequence included two of the three sites that make up the catalytic triad, and the respective amino acids were 100% conserved. The deduced partial protein further contained a predicted lipid-binding site with extremely high conservation (15/18 residues) compared with the human ortholog, and it retained the proposed apoCII binding site (Lys-196 in zebrafish). Using neighbor-joining phylogenetic analysis (data not shown), homology with LPL orthologs proved higher than that with other lipases. Sequence identity ranged from 82% in teleost fish, such as rainbow trout (Oncorhynchus mykiss; accession no. AAK69707) and zebrafish (NP_571202) to around 70% in mammals (e.g., human AAH11353 and rat NP_036730).Experiment 1: Lipid content, LPL mRNA levels, and LPL enzyme activity in the ovary and liver of wild shortfinned eelsOvary.With the initiation of puberty, the ovary increased in relative size (GSI) from 0.2 ± 0.03% in PV eels in the chromatin nucleolus or early oil droplet stage to 3.1 ± 0.11% (P < 0.01) in EV eels. Over the same period, the lipid content of the whole ovary increased approximately 100-fold (t =−14.94, df = 7.9, P < 0.05) in the EV ovary (Fig. 1A) of eels that were of standardized, equal size (i.e., AEM-corrected). This dramatic increase in ovarian lipid content coincided with a significant (t =−10.33, df = 12.8, P < 0.05) 30-fold increase in the whole-organ ovarian LPL mRNA copy number by the time vitellogenesis had started (Fig. 2A). When the LPL mRNA copy number was expressed per mg ovary, this increase remained significant, although the magnitude of the change (2-fold) was much lower (data not shown).Fig. 2Total ovarian mRNA copy number of lipoprotein lipase (LPL) in eel, Anguilla australis, at different stages of reproductive development. Eels were either collected from the wild (A) or repeatedly treated with salmon pituitary homogenate or Ringer solvent (CTRL, Ringer solvent controls; sampled at different time points, but analyzed collectively as controls did not differ over time) to induce gonadal development (B). Data are standardized for body length using an allometric correction (BL2.91, body length; see text for details). All data are presented as means ± standard error and sample sizes are given at the base of each bar. Different letters above bars indicate significantly different treatment means (P < 0.05). Asterisks indicate that differences are significant at P < 0.001. Abbreviations: EV, early vitellogenic after 2 (EV2) or 4 (EV4) weeks of pituitary homogenate treatment; MV, midvitellogenic; P-OV, postovulatory ovary; PV, previtellogenic.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Total LPL activity in the ovary displayed a profile with developmental stages very similar to lipid content and transcript levels, LPL activity being about 30-fold greater (t =−4.67, df = 6, P = 0.003) in wild EV eels (98.29 ± 42.22 mU LPL/BL2.91) than in PV eels (3.1 ± 1.43 mU LPL/BL2.91) (Fig. 3A). This large increase reflects both increases in organ size and absolute activity, as seen by the 3-fold increase in LPL activity expressed per