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BOR - Papers in Press, published online ahead of print March 22, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049494
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BIOLOGY OF REPRODUCTION 75, 34–44 (2006)
DOI: 10.1095/biolreprod.105.049494
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Previtellogenic Oocyte Growth in Salmon: Relationships among Body Growth, Plasma Insulin-Like Growth Factor-1, Estradiol-17beta, Follicle-Stimulating Hormone and Expression of Ovarian Genes for Insulin-Like Growth Factors, Steroidogenic-Acute Regulatory Protein and Receptors for Gonadotropins, Growth Hormone, and Somatolactin1

B. Campbell 5, J. Dickey 5, B. Beckman 6, G. Young 57 , A. Pierce 3 5, H. Fukada 4 6, and P. Swanson 2 67 

School of Aquatic and Fishery Sciences,5 University of Washington, Seattle, Washington 98195 Northwest Fisheries Science Center,6 National Oceanic and Atmospheric Administration-National Marine Fisheries Service, Seattle, Washington 98112 Center of Reproductive Biology,7 Washington State University, Pullman, Washington 98164

ABSTRACT

Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

estradiol, follicle-stimulating hormone, follicle-stimulating hormone receptor, growth hormone, oocyte development

INTRODUCTION

Female salmon (Oncorhynchus species) native to the Pacific Northwest coast of North America spawn only once in a lifetime and exhibit a large variation in the age of maturity, as well as body size, egg size, and fecundity [1]. Although genetic factors underlie this phenotypic plasticity, environmental factors that influence growth during critical periods of the life cycle play important roles. For example, in autumn-spawning salmonids rapid growth in the fall 1 yr before spawning affects initiation of maturation, whereas rapid growth through a subsequent permissive period is required for maturation to continue [2–7]. Furthermore, growth during early stages of oogenesis affects egg size and fecundity [8, 9] suggesting that body growth significantly affects critical transitions in oogenesis, such as recruitment of oocytes into vitellogenesis, as well as oocyte growth and survival. Although interactions between the growth and reproductive systems in female fish are profound, the underlying physiological mechanisms are poorly understood.

Substantial efforts to determine the mechanisms regulating vitellogenic growth and final oocyte maturation in teleosts have occurred in recent years [10]. However, the factors controlling primary and early secondary oocyte growth are poorly understood. During primary growth, oocytes arrest in prophase I of meiosis, and the classic ovarian follicle structure is formed with perinucleolar oocytes surrounded by granulosa and thecal cells. Before yolk incorporation, the follicle undergoes a secondary growth phase with the sequential accumulation of cortical alveoli (the cortical alveolus stage) and lipid droplets (the lipid droplet stage) in the ooplasm and formation of an acellular layer (the zona radiata) between the ooplasm and granulosa cell layer [11]. In salmonids the timing of early stages of oocyte development before onset of yolk incorporation coincides with periods during which growth affects maturation. Thus, the early previtellogenic growth period is central in determining the age of maturation, fecundity, and egg size in salmon and probably other fish species.

In mammals ovarian development is driven by a combination of systemic and intraovarian factors, the relative importance of which depends on the stage of oocytes [12–14]. It is now apparent that similar systems are present in teleost fish [15–17]. Gonadotropins (FSH and LH) promote follicular steroid production and oocyte growth; however, the relative roles of FSH and LH in vitellogenesis and oocyte maturation may differ among species [18, 19]. In salmonids studies suggest that secondary oocyte growth is regulated primarily by FSH, whereas LH plays a major role in regulating final oocyte maturation and ovulation. Evidence from a variety of fish species indicates that the growth hormone-insulin-like growth factor system (hereafter "GH-IGF," when referring to this system; otherwise, the nomenclature for each separate gene follows standards specified in appropriate gene nomenclature databases) and somatolactin (hereafter "Sl," for consistency with the nomenclature for its receptor in the ZFIN database) also may play important roles in regulating ovarian development. Ovarian steroidogenesis is stimulated by growth hormone (Gh) [20, 21], Sl [22], and insulin-like growth factor 1 (Igf1) [23–25]. In a number of fish species, Igf1 either induces competence of the oocyte to respond to maturation-inducing steroids, or it induces final oocyte maturation directly [26–30]. Transcripts for igf1 and igf2 and receptors for Gh (ghr), Sl (slr), Igf1, and Igf2 are expressed in the ovarian follicle [31–36]. However, the precise roles that FSH, sex steroids, Sl, and the GH-IGF system play in regulating primary and early secondary oocyte growth in fish have yet to be defined.

The aims of this study were to characterize previtellogenic oocyte growth in relation to age and body growth in coho salmon (Oncorhynchus kisutch). This species has a simple life history and generally spawns in the late fall at the age of 3 yr, unless growth is altered. Changes in components of the FSH-ovary axis and intraovarian GH-IGF system were monitored during periods hypothesized to be important for the direction of oocyte development. Ovarian slr expression was also monitored to obtain basic information on its relation to ovarian development. Plasma levels of Sl increase during early stages of oocyte growth in salmon [37, 38], suggesting that Sl may play an important role in oocyte recruitment. Transcripts for steroidogenic acute regulatory protein (star) were measured because of its role in regulating cholesterol transport into the mitochondria, which is a critical rate-limiting step in steroid biosynthesis [39]. Studies in mammals suggest that both gonadotropins and IGF1 regulate steroidogenesis in part by stimulating STAR gene expression [40].

We hypothesized that, in salmon, the two critical periods during which growth influences previtellogenic ovarian development are the fall and the spring before spawning; that the degree of advancement in oocyte development by the spring before spawning is related to body growth before that point; that plasma Igf1 is a critical growth factor that signals growth and nutritional status to the reproductive axis, thereby influencing oocyte development; and that transitions in previtellogenic oocyte growth are associated with changes in FSH signaling, the intraovarian GH-IGF system, and steroid biosynthesis. Therefore, changes in ovarian gene expression and plasma hormone levels were monitored during the fall critical period (from August through December of the second year of age) and at one point during the following spring (in May of the third year of age).

MATERIALS AND METHODS

Fish, Rearing Conditions, Diets, and Feeding

Yearling coho salmon (1999 brood, University of Washington stock) were reared at the Northwest Fisheries Science Center hatchery facilities (Seattle, WA) in duplicate 2.4-m diameter fiberglass tanks with recirculated fresh water (10–11°C). In February 2001, a total of 1300 fish (≥1 yr old) were individually tagged with passive-integrated transponder (PIT) tags, and fin tissue was collected from all fish for determination of genetic sex by detection of a molecular marker for the Y chromosome [41]. The genetic sex identification was necessary so that the majority of males could be removed early in the experiment because of rearing space limitations. After tagging, fish were divided into four replicate tanks. A daily ration was calculated using the growth model based on the delta-l method of Piper et al. [42] to produce a mean fish weight of approximately 33 g by May 2001. Fish were fed a commercially available diet (BioDiet Grower; BioOregon, Warrenton, OR).

In May 2001, after the transformation of juvenile parr into smolts (the migratory stage), fish were sorted by sex, and all 620 females and 100 males were transported to the National Marine Fisheries Service Manchester Marine Field Research Station (Manchester, WA). Fish were randomly split into four 3.6-m diameter fiberglass tanks supplied with filtered and UV-treated seawater. Temperature varied seasonally from 8–14°C. A daily ration was calculated (as determined above) to produce a mean fish weight of approximately 230 g by May 2002. Fish were fed a commercially available diet (BroodSelect; Skretting, Vancouver, BC). As is common in coho salmon, a portion of the fish transferred to seawater became stunts and grew poorly or died after transfer [43]. All such animals that remained in August were culled from the population by use of a conservative size criteria (body weight of <30 g) and observation of body morphology typical of stunted growth (i.e., large head, emaciated body, and listlessness). Fish were given intraperitoneal injections of oxytetracycline (30 mg/kg body weight) during November 2001 and January 2002 to reduce the risk of bacterial infection. All females showing clinical signs of infection were culled during monthly sampling. Only healthy females were used for experimental sampling. In May 2002, six months before spawning, all females were terminally sampled to assess the degree of ovarian growth.

Animal use procedures followed the policies and guidelines of the University of Washington Institutional Animal Care and Use Committee (IACUC 2313–09).

Sample Collection, Hormone Analyses, and Histology

Body weight and length were recorded monthly for all fish from January 2001 through January 2002 and at the final terminal sample in May 2002. A total of 14–19 fish/mo were terminally sampled from the rearing tanks from August 2001 through December 2001. The remaining 73 females (16–20 fish/tank) were terminally sampled in May 2002. The timing of sampling relative to the coho salmon life cycle is illustrated in Figure 1. The gonadosomatic index (GSI) of individual fish was calculated as [ovary weight/body weight] x 100. Tissue and blood specimens were collected from fish at each sampling date for measurement of pituitary and plasma hormone levels, for measurement of transcript levels in the ovaries, and for histologic analysis. Fish were killed in buffered tricaine methanesulfonate (0.05% MS-222; Argent Chemical, Redmond, WA), and body weight and length were recorded. Blood was collected via the caudal vein, using heparinized syringes and 21-gauge needles. Pituitary specimens were frozen in liquid nitrogen and stored at –70°C. Ovaries were weighed, and two specimens were collected; one was frozen in liquid nitrogen (for mRNA analyses, see below), and the other was fixed in Bouin fixative for 24 hr before storage in 70% ethanol. Fixed ovaries were dehydrated through a graded series of ethanol and embedded in paraffin wax, sectioned (thickness, 4 µm), and stained with hematoxylin-eosin. Stages of oogenesis were determined by light microscopy, using the protocol of Nagahama [11] as a guide. Blood was centrifuged at 1000 x g for 5 min, and plasma was stored at –70°C. Plasma estradiol-17ß (E2) was measured directly by radioimmunoassay with a commercially available kit (ICN Diagnostic Products, Irvine, CA). Serial dilutions of plasma were parallel to the standard curve, and the intraassay and interassay coefficients of variation were 4.5% and 13.1%, respectively. The plasma Igf1 concentration was measured by radioimmunoassay with commercially available components (GroPep, Adelaide, Australia) as described by Shimizu et al. [44] only for samples obtained in May, because of insufficient plasma volume from samples obtained during the fall. The intraassay and interassay coefficients of variation were 1.4% and 8.7%, respectively. Pituitary FSH and plasma FSH levels were measured by radioimmunoassay as described previously [45, 46]. The intraassay and interassay coefficients of variation were 1.8% and 12.5%, respectively, and the sensitivity of the assay after a 72-hr primary antibody incubation was 0.16 ng/ml.


Figure 1
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  		FIG. 1. The timing of sampling relative to the coho salmon life cycle.

RNA Isolation and Reverse Transcription (RT) for Real-Time Quantitative RT-PCR Assays

Total RNA was isolated from frozen tissue specimens by means of TRI REAGENT (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Integrity of the RNA was verified by an optical density (OD) absorption ratio (OD 260 nm/OD 280 nm) of >1.9 and quantified by spectrophotometry at 260 nm (GeneQuant; Pharmacia Biotech, Cambridge, England). Total RNA was diluted in 5 ng/µl nuclease-free water, and 15 µl was transcribed using Superscript II RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA). RT-PCR reaction conditions were as follows: 3.0 µl of 5x buffer, 1.5 µl of 0.1 M dTT, 0.75 µl of dNTPs (stock of 10 mM each of dCTP, dGTP, dTTP, and dATP; Promega, Madison, WI), 0.225 µl of random hexamer (500 ng/µl stock; Promega), 0.1875 µl of Superscript II RNase H-RT (200 U/µl), 0.3 µl of RNase inhibitor (20 U/µl; Applied Biosystems [ABI], Foster City, CA), sterile distilled deionized (dd) H2O (6.0375 µl), and 3.0 µl of template. The temperature profile for the RT-PCR was as follows: 25°C for 10 min, 48°C for 60 min, and 95°C for 10 min, followed by a 4°C incubation.

Determination of Partial cDNA Sequences of Coho Salmon FSH Receptor (fshr), LH Receptor (lhcgr), star, and igf2

Coho salmon cDNA sequences for fshr, lhcgr, star, and igf2 were determined to permit design of primers and probes for real-time quantitative RT-PCR assays. For fshr and lhcgr, total RNA was isolated from deyolked follicles from vitellogenic coho salmon ovaries and reverse transcribed as described above, except that 4.4 µg of total RNA and 500 ng of oligo dT primer were used for the RT reaction. Nested PCR was performed with primers designed from amago salmon (O. rhodurus) fshr (GenBank accession no. AB030012) and lhcgr (GenBank accession no. AB030005) cDNA sequences (Table 1). The first round of PCR was performed in 50 µl with first strand cDNA as a template and a GeneAmp PCR System 2400 (Perkin Elmer) under the following conditions: 94°C for 2 min, and 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min. Final extension of PCR products was 72°C for 7 min. After electrophoresis in 1.2% agarose gel, multiple bands were observed, and the brightest band of expected size was excised from the gel and purified using a Qiaex II Gel Extraction Kit (Qiagen, Valencia, CA). The second round of PCR (50 µl) was performed on each respective purified band (2.0 µl of a 1:10 dilution) with nested primers under the same PCR conditions from the first PCR except a reduction in cycle number to 25. After electrophoresis, a single band of expected size was purified and sequenced using Big Dye Terminator Cycle Sequencing Kit and an ABI PRISM 3100 Genetic Analyzer.


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  		TABLE 1. Primer sequences for PCR cloning.

For igf2, total RNA was isolated from coho salmon liver, and RT-PCR was performed as above using 1.0 µg of total RNA and primers designed to conserved regions of rainbow trout (O. mykiss; GenBank accession no. AB047032) and chum salmon (O. keta; GenBank accession no. X97225) igf2 cDNA (Table 1). After electrophoresis, a single band of expected size was purified and sequenced as described above for fshr and lhcgr.

For star, total RNA was isolated from coho salmon head kidney/interrenal tissue by using a GeneChoice RNA Spin Mini Kit (PGC Scientific, Gaithersburg, MD). RT-PCR was performed using 2.0 µg total RNA and GeneChoice's Thermo RT II One Tube Kit, in accordance with the manufacturer's instructions, and with primers that were designed to conserved regions of star from rainbow trout (GenBank accession no. AB047032) and brook trout (Salvelinus fontinalis; GenBank accession no. AF232215) (Table 1). Conditions for RT-PCR were as described above for fshr and lhcgr. After electrophoresis, a single band of expected size was observed, excised, and ligated into the TOPO cloning vector using TOPO TA Cloning kit (Invitrogen). Positive clones were color selected, screened by PCR, and sequenced on an ABI PRISM 310 Genetic Analyzer with the ABI Big Dye Terminator Cycle Sequencing Kit v2. All cDNA sequences obtained for coho salmon igf2, star, fshr, and lhcgr had 97%–99% base pair identity with the respective cDNAs from other salmonid species used for primer design.

Real-Time Quantitative RT-PCR Assays

Probes and primers for real-time quantitative RT-PCR assays were designed according to sequence data by means of Primer Express software (ABI). When possible, intron/exon splice junctions were used in primer design to avoid potential signal contamination by genomic DNA (Table 2). For each transcript that was measured, a total RNA sample was analyzed without RT reaction to test for DNA contamination. Assays for ghr, slr, and igf1 were developed for coho salmon and published elsewhere [35, 36, 47]. All assays were run on an ABI 7700 Sequence Detector in 96-well plates and ABI's Universal PCR MasterMix Reagent. PCR efficiency for each transcript was measured using a serial dilution of an ovarian total RNA sample from within the experiment as a reference. Standard-curve dilutions were run in triplicate, whereas analysis of samples was not replicated. Reaction conditions were as follows for 25-µl PCRs: 12.5 µl of Universal PCR MasterMix (ABI), 0.5 µl of forward primer (45-µM stock), 0.5 µl of reverse primer (45-µM stock), 0.5 µl of probe (10-µM stock), 8.0 µl dd H2O, and 3.0 µl of RT reaction. Cycling parameters were as follows: 50°C for 2 min, 95°C for 10 min, and 40–45 cycles of 95°C for 15 sec followed by 60°C for 1 min. Transcript levels were calculated using the serially diluted total RNA sample standard curve and were expressed relative to the amount of reverse-transcribed template RNA that went into the real-time PCR. Template RNA concentrations were quantified using a NanoDrop ND-100 (NanoDrop Technologies, Wilmington, DE), and the samples were diluted to the same concentration before the RT reaction. This method of calculation was performed because significant variation was observed between oocyte stages for housekeeping gene transcripts (18S, acidic ribosomal protein, and elongation factor alpha). All samples within one set of comparisons (August through December 2001 or May 2002) were contained within one plate. The relative level of gene expression was calculated by dividing the above values by the mean of a designated group; for the fall samples (August through December 2001) transcript levels in August 2001 were used, and for the May (2002) samples the CA-group were used (see below). This manipulation provides a clear representation of the relative changes in expression of each gene over time in the fall or between groups in May.


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  		TABLE 2. Primer and probe sequences for TaqMan RT-PCR assays.

Statistical Analysis

Data were log transformed, and percentage data were arcsine transformed to meet parametric test criteria. Differences between fish in each tank at each sample date were determined by analysis of variance (ANOVA). Because no tank-based differences were found, data from replicate tanks were combined. Significant differences among sample dates during fall 2001 were determined by ANOVA, followed by Tukey multiple mean comparison tests.

For the data from the May 2002 samples, fish were separated into two groups according to results of ovarian histologic analysis. Ovaries were classified into one of two stages: cortical alveoli (CA) or lipid droplet (LD). Levels of all factors measured were compared between these two groups by means of the Student t-test. Correlations (correlation coefficient, r) between parameters were calculated by means of the Pearson K test. Forward stepwise multiple regression analysis of plasma E2 in May on the other parameters measured was performed to determine the relationships between each of these factors and plasma E2 levels. Oocyte staging was included in the analyses as a nominal factor. The P value for entry into the model was set at 0.05. Comparison of growth history between the females from the CA group and LD group in May was performed by comparing the difference in body weight between the two groups at each date using Student t-test. The significance level was adjusted by the Bonferroni correction to reduce type I error because multiple Student t-tests were used. All analyses were performed using JMP (SAS, Carey, NC). Data in the figures are mean ± SEM. The level of statistical significance was set at P < 0.05.

RESULTS

Fall

In August 2001 the stage of ovarian development varied among females, ranging from the late perinucleolar to the early cortical alveolus stage. By December all ovaries contained oocytes with cortical alveoli (intermediate between early and late cortical alveolus stage); none remained at the late perinucleolar stage. Follicle morphologic characteristics and relative sizes for various oocyte stages are illustrated in Figure 2. Body weight and GSI (Fig. 3) increased significantly during the fall of 2001. Plasma E2, FSH, and pituitary FSH levels also increased significantly during the fall (Fig. 3). Ovarian star transcript levels increased from August to December, whereas ghr, slr, and lhcgr transcript levels significantly decreased (Fig. 4). Levels of igf1 transcripts in the ovary had a distinct peak during September, whereas igf2 transcript levels decreased significantly between September and December. Throughout the fall, levels of fshr transcript remained unchanged.


Figure 2
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  		FIG. 2. Morphology and relative sizes of the perinucleolar, early cortical alveolus, late cortical alveolus, and lipid droplet stages of oocyte development in coho salmon. Arrow indicates centrally located lipid droplet. Bar = 100 µm.


Figure 3
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  		FIG. 3. Changes in body weight, GSI, pituitary FSH, plasma FSH, and plasma E2 for the female coho salmon sampled during the fall (between August 2001 and December 2001), 1 yr before spawning. Bars with similar letters are not significantly different. Data are presented as mean ± SEM (n = 14–19 fish/mo).


Figure 4
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  		FIG. 4. Relative changes in gene expression of ovarian fshr, lhcgr, ghr, slr, igf1, igf2, and star for coho salmon sampled during the fall (between August 2001 and December 2001), 1 yr before spawning. Levels are expressed as a proportion of the mean value in August 2001. Bars with similar letters are not significantly different. Data are presented as mean ± SEM (n = 14–19 fish/mo).

Body weight and ovary weight both positively correlated with plasma E2 and pituitary FSH levels, but only ovary weight positively correlated with levels of plasma FSH (Table 3). Plasma E2 levels were positively correlated with plasma FSH and pituitary FSH levels, which were positively correlated with each other. Relative changes in star expression were positively correlated with those of fshr and with body weight and plasma E2 levels. Ovary weight and levels of plasma FSH and pituitary FSH were negatively correlated with relative changes in lhcgr transcript levels, and changes in fshr transcript levels were negatively correlated with plasma FSH levels. Other correlations were not significant. Thus, there was a general interrelationship among the plasma and pituitary components of the reproductive axis. Expression of star was the only ovarian gene that was positively correlated with any of these extraovarian factors.


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  		TABLE 3. Correlation coefficients (r) among reproductive parameters from samples collected during the fall (top value, n = 64) and May.*

Spring

In May 2002 females could be divided into two distinct groups on the basis of the stage of oocyte development: females with oocytes with large centrally located lipid droplets (designated as the LD group) and females with oocytes in varying degrees of cortical alveoli incorporation and no lipid droplets (designated the CA group). The growth histories of females in the LD group significantly diverged from those of females in the CA group during September 2001, with less growth in the CA group from this point up to May 2002 (Fig. 5). By the end of the experiment, fish from the LD group were twice the size of fish in the CA group.


Figure 5
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  		FIG. 5. Growth history of the CA (cortical alveolus, open circles) and LD (lipid droplet, closed squares) groups of coho salmon females terminally sampled in May 2002. Stars indicate a significant difference in the body weight between the two groups of fish. Data are presented as mean ± SEM (n = 31–33 fish/mo). Shaded areas indicate hypothetical critical periods for ovarian development in the fall and spring. Arrow indicates the expected time of spawning for the LD group (from November through December 2002).

Potential physiological differences between the CA and LD groups in May 2002 were assessed by comparing all the factors measured (Figs. 6 and 7). Body size and GSI in the LD group were significantly higher than those in the CA group (Fig. 6). Similarly, plasma E2, FSH, Igf1, and pituitary FSH levels in the LD group were significantly higher than those in the CA group in May (Fig. 6). In the ovary, fshr, lhcgr, star, igf1, and igf2 transcript levels in the LD group were significantly higher than those in the CA group (Fig. 7). In contrast, slr and ghr transcript levels in the LD group were significantly lower than those in the CA group.


Figure 6
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  		FIG. 6. Body weight, GSI, pituitary FSH, plasma FSH, plasma E2, and plasma Igf1 for fish in the CA (cortical alveolus; n = 31) and LD (lipid droplet; n = 33) groups sampled in May 2002. Bars with similar letters are not significantly different. Data are presented as mean ± SEM.


Figure 7
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  		FIG. 7. Relative changes in gene expression of ovarian fshr, lhcgr, ghr, slr, igf1, igf2, and star for fish in the CA (cortical alveolus; n = 31) and LD (lipid droplet; n = 33) groups sampled in May 2002. Levels are expressed as a proportion of the mean value in the CA group. Bars with similar letters are not significantly different. Data are presented as mean ± SEM.

Correlations among body growth and factors in the gonadotropin-ovary axis. In the CA group in May, body weight and ovary weight were both positively correlated with levels of plasma E2 and pituitary FSH (Table 3). Plasma E2 levels were positively correlated with levels of plasma FSH and pituitary FSH, which were positively correlated with each other. Relative changes in star expression were positively correlated with those of fshr and plasma FSH levels. Relative changes in expression of ovarian fshr and lhcgr were also positively correlated. Other correlations were not significant. Thus, as in the fall, there was a general interrelationship among the plasma and pituitary components of the reproductive axis. The star gene was the only ovarian gene that was positively correlated with any of these extraovarian factors.

In the LD group in May, body weight and ovary weight were both positively correlated with plasma E2 levels, but only body weight was correlated with pituitary FSH levels. Plasma E2 and pituitary FSH levels were positively correlated, but plasma FSH levels were not significantly correlated with either of these factors. The relative changes in star transcripts were positively correlated with body weight and plasma E2 levels. Other correlations were not significant. Thus, the relationship between plasma E2 and pituitary FSH levels was consistent with that in the CA group, but plasma FSH levels were not significant related to extraovarian factors in the LD group. The star gene remained the only ovarian gene whose change in expression was correlated with any of these extraovarian factors.

Correlations among body growth, plasma Igf1 levels, and intraovarian ghr, slr, igf1, and igf2 levels. In the CA group in May, plasma Igf1 levels were positively correlated with body weight and ovary weight, but they were not correlated with changes in ovarian ghr, slr, or igf expression (Table 4). Relative changes in ghr and slr transcripts were both negatively correlated with body weight, but only changes in ghr were negatively correlated with ovary weight. In the ovary, changes in ghr expression were positively correlated with those of slr, whereas changes in igf1 transcript levels were positively correlated with changes in igf2. Thus, there was a general positive relationship among plasma Igf1 levels, body size, and ovary size but a negative relationship between body size or ovary growth and changes in expression of ghr and slr.


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  		TABLE 4. Correlation coefficients (r) between growth parameters and intraovarian growth factors from samples collected during May at the cortical alveolus (n = 31, upper value) and lipid droplet (n = 33, lower value) stages of oocyte development.

In the LD group in May, plasma Igf1 levels were positively correlated with body weight, but they were not correlated with changes in ovarian growth-factor transcripts. Changes in ovarian ghr transcripts positively correlated with those of slr, whereas changes in igf1 transcripts were positively correlated with those of igf2. The relative changes in ghr expression were negatively correlated with those of igf1 but positively correlated with changes in igf2 expression. Thus, there was a general positive relationship between plasma Igf1 levels and body size but no relationship with ovarian growth-factor transcripts.

Relationship between plasma Igf1 levels and reproductive factors in the spring. In the CA group, plasma Igf1 levels correlated positively with levels of plasma E2, pituitary FSH, and plasma FSH but not with any of the ovarian transcripts (Table 3). In the LD group, plasma Igf1 levels correlated positively with levels of plasma E2, levels of pituitary FSH, and changes in star expression but not with plasma FSH levels or the other ovarian transcripts.

Forward stepwise regression was performed to determine which factors were the best predictors of plasma E2 levels in May, with oocyte stage (CA-LD) as a nominal factor. Body size was the main predictor of plasma E2 levels (r2 = 0.72). Plasma Igf1 levels next entered the regression model, increasing the relationship by 8% (r2 = 0.80). Oocyte stage and changes in ovarian fshr transcript levels contributed a further 3% to the model (final model, r2 = 0.83; P < 0.0001). None of the other parameters were significant predictors of plasma E2 levels.

DISCUSSION

The aims of this study were to investigate the relationship between previtellogenic oocyte growth and body growth in coho salmon and to characterize the associated changes in the gonadotropin-ovary axis and intraovarian GH-IGF system. Previous studies suggested that body growth in salmonids during critical periods affects the proportion of fish that will mature in a given year [2–7]. A model based on these studies proposed that two critical periods exist, one for initiation of maturation in the fall and another for permitting maturation to continue in the spring [3]. Thus, the present study focused on the fall and spring critical periods. Our findings confirm that body growth through the fall and winter months profoundly affects ovarian growth before onset of vitellogenesis (i.e., the transition from the cortical alveolus to the lipid droplet stage). We provide the first physiological evidence to support the contention that, for a fall spawning salmonid, such as coho salmon, an initiation period for ovarian maturation occurs in the fall, 1 yr before spawning. Furthermore, our data suggest roles for Igf1, FSH, E2, and intraovarian genes in the recruitment of oocytes into secondary oocyte growth, which may be analogous to follicular selection in mammals [48].

Body Growth and Ovarian Development

In this study, the degree of advancement of follicular development from the fall through the spring was related to body growth. At the first sampling in August, females had ovaries with oocytes that ranged from the perinucleolar stage to very early cortical alveolus stage. During the subsequent fall period (August through December) ovary mass increased, and oocytes accumulated cortical alveoli in the ooplasm. By May fish diverged into two groups. The CA group remained in the cortical aveolus stage, and the LD group had oocytes that had advanced to the lipid droplet stage. A review of the growth history of the fish sampled in May indicated that females in the CA group had significantly less body growth than females in the LD group from the previous September onwards. It is clear that body growth during the late fall and winter months was related to the degree of oocyte development during the spring, with larger fish being more advanced than smaller, slower-growing fish. On the basis of the present data, it is not known whether fish in the CA group would have ultimately spawned in November. However, previous work in individually tagged coho salmon demonstrated that females as small as those in the CA group in May remained immature, whereas those similar in size to fish in the LD group matured and spawned in the fall (unpublished results). It is likely that this relationship between body growth and oocyte development dictates whether a critical stage in oocyte development is reached during the spring and determines whether the ovarian follicle then subsequently develops to the point of ovulation in the fall. Unfortunately, because of the low number of terminal samples collected in the fall, we could not detect bimodality in oocyte stage or reproductive hormones in relation to growth. Thus, it remains to be determined whether a true split in reproductive physiology due to body growth occurs during the fall, 1 yr before ovulation.

Gonadotropin-Ovary Axis

Accumulation of cortical alveoli. During the fall, 1 yr before spawning, the accumulation of cortical alveoli in the ooplasm and increases in ovary mass were associated with increases in pituitary FSH, plasma FSH, plasma E2, and ovarian star transcript levels. The positive correlation among these factors in fish during the fall and in fish at the cortical alveolus stage in May provide evidence for an interrelationship between these factors during this period of oocyte development. These data suggest that FSH and E2 may be involved in the regulation of oocyte development during the accumulation of cortical alveoli.

It is well established in mammalian models that FSH is obligatory for antral follicle growth, because the ablation of FSH action or receptor expression results in arrest of follicle growth. Before this stage, FSH is not obligatory but stimulates follicular development [14, 49]. Selection of the follicles for subsequent ovulation is largely dependent on FSH [48]. Recent work in channel catfish (Ictalurus punctatus) and zebrafish (Danio rerio) has suggested a rise in pituitary fshb ( follicle stimulating hormone, beta polypeptide), and ovarian fshr gene expression occurs prior to vitellogenesis, coinciding with the accumulation of cortical alveoli, and this upregulation continues through vitellogenesis [50–52]. In the present study ovarian fshr transcript levels did not increase in parallel with pituitary and plasma FSH levels during the fall; however, plasma FSH levels and relative changes in star expression were correlated with expression of fshr, suggesting that FSH signaling is important during the accumulation of cortical alveoli.

The role of LH in the cortical alveolus stage is unclear. Transcripts for lhcgr were detectable (albeit at low levels, as indicated by high CT values), and expression decreased during the fall, with no clear relationship to other factors. In salmonids, FSH is associated with early stages of oocyte development, whereas LH levels are low or undetectable until final oocyte maturation and ovulation [45, 53–56]. Thus, in salmonids, it is generally thought that FSH plays the major role in follicular growth and development. The role of Lhcgr in mediating the actions of FSH during previtellogenic oocyte growth in salmon is not clear, because there are conflicting reports on whether FSH can interact with the Lhcgr [57–60] and because LH is not detectable in the circulation at this time. Recent studies suggest that gonadotropin subunits are produced within the ovary of teleosts [61], raising the possibility that LH within the ovary could act as the major ligand for Lhcgr at this stage.

Similar to mammals, the major actions of gonadotropins in teleosts are mediated through steroid production. FSH and E2 levels increase together during maturation [45, 53, 54], and FSH stimulates E2 production and alters ovarian aromatase activity [62–64]. In addition to stimulating hepatic vitellogenin production [65], E2 has feedback actions on GnRH and gonadotropin production and release [19]. Intraovarian actions of E2 are less well characterized in fish.

In mammals, it is known that E2 also has intraovarian actions: it interacts with FSH and IGF1 to stimulate proliferation and differentiation of follicular cells. Gene knock out of E2 receptor and aromatase in mice arrests follicular development at the preantral or early antral stages of follicular development [66]. The recent identification of E2 receptors in the salmonid ovary may indicate that a similar system operates in fish [67]. E2 has also been implicated in both cortical alveoli incorporation and the production of zona radiata proteins (zonagenesis) in teleosts [68, 69]. In the present study, the increase in ovarian star transcript and plasma E2 levels during the fall supports our contention that E2 plays important roles during cortical alveoli deposition long before significant amounts of vitellogenin are produced in the liver.

Accumulation of lipid droplets. The accumulation of lipid droplets in females in May was associated with increases in levels of pituitary and plasma FSH, plasma E2, and ovarian star, fshr, and lhcgr transcripts, compared with females who remained in the cortical alveolus stage. Increases in transcripts for pituitary fshb and ovarian fshr before onset of vitellogenesis have been reported in other species [50–52]. In striped bass, increases in pituitary fshb transcripts were reported with the appearance of lipid droplets in the oocyte [70, 71]. The general increase in components of the gonadotropin system as lipid droplets are deposited indicates an upregulation of this system; however, in the present study plasma E2 levels positively correlated with pituitary FSH and changes in star expression and not with plasma FSH levels. It is possible that other factors in addition to FSH may play important roles in regulating production of E2 at this stage (see below). Furthermore, expression of fshr and of plasma and pituitary FSH levels in the LD group were all significantly higher than those in the CA group. This suggests that FSH signaling is probably important for the transition into the lipid droplet stage but that its effect may not occur via effects on star expression or steroid biosynthesis. Although lhcgr expression was higher in follicles in the lipid droplet stage, the role of lhcgr signaling in accumulation of lipid droplets and steroid biosynthesis is unclear, because of the lack of detectable LH in plasma at this stage.

Intraovarian GH-IGF System

In the fall, as FSH and E2 levels increased, gene expression of the components of the intragonadal GH-IGF system decreased, corroborating previous research indicating that ghr, igf1, and igf2 gene expression were highest in the nucleus and cytoplasm of oocytes in the chromatin-nucleolus and perinucleolus stages in gilthead seabream and tilapia [31, 34]. This suggests that Gh, Sl, or Igfs may play more significant roles in earlier stages of primary oocyte growth, but the data do not preclude roles for these factors in later stages of oocyte development, when transcripts are still present. In seabream and tilapia, ghr, igf1, igf1r, and igf2 transcripts were detected in the granulosa and theca cells in vitellogenic follicles, suggesting a role for these factors in later oocyte development. Igfs, Gh, and Sl have all been shown to stimulate steroidogenesis in the ovary [20–25], and plasma levels and gene expression increase during maturation [32, 33, 38, 72, 73]. However, there is little evidence from this study or previous work to suggest that Gh or Sl have major roles in cortical alveoli or lipid droplet accumulation in salmonids. The potency of Gh and Sl in stimulating steroidogenesis is far less than that of gonadotropins [20–22], and the expression of ghr and slr appeared to decrease as the oocyte progressed through early secondary growth. Indeed, Ghr knock out female mice still mature and produce fertile oocytes, but at a reduced rate [74]. Thus, GH (and Sl in fish) may have more of a facilitatory rather than obligatory role in oocyte development.

In the present study ovarian igf1 and igf2 expression was higher in ovaries that had oocytes in the lipid droplet stage, suggesting that these factors may play a role in oocyte development during early secondary oocyte growth. The intraovarian Igfs in fish may be important for maintaining oocyte development, regulating gonadotropin receptors, and promoting steroidogenesis, as in mammals [74]. At this point, the role of paracrine versus endocrine Igfs in ovarian growth in fish has not been investigated.

Role of Plasma Igf1

Plasma Igf1 is a principal component of the growth axis and is often highly correlated with growth rate and body size in salmonids [75–77]. Increases in plasma Igf1 that occur with increased growth rate and body size in fish may be one of the key components activating the reproductive axis. In the present study, plasma Igf1 was significantly correlated with body size during May, and both factors were significantly higher in fish that had commenced the incorporation of lipid droplets than in fish remaining in the cortical alveolus stage. We hypothesize that, during spring, the growth-associated increase in plasma Igf1 augments the production of factors associated with ovary growth and that E2 is a major factor involved in the cross talk between the somatotropic and reproductive axes. Plasma Igf1 and E2 levels were consistently positively correlated, and stepwise multiple regression analysis of the relationship between plasma E2 levels and all the other parameters measured suggested that body size and plasma Igf1 were important factors influencing plasma E2 levels. Igf1 can have direct effects on steroidogenesis by stimulating aromatase [25] and E2 production [24]. In mammals, IGF1 and FSH stimulate expression of STAR and other steroidogenic enzyme genes [40]. In the present study, plasma Igf1 and E2 levels were correlated with changes in star expression in fish incorporating lipid droplets, which supports the contention that Igf1, E2, and star interact during this stage of oocyte development. Endocrine Igf1 may either stimulate star expression directly or act synergistically with FSH [40], resulting in increased steroid biosynthesis. However, it should be noted that intraovarian Igfs may also play a similar role, because the relative expression of intraovarian igf1 and igf2 increased as lipid droplets appeared. In addition to effects on ovarian physiology, Igf1 can increase pituitary FSH production and secretion in response to GnRH in fish [78, 79], and in mammals E2 and IGF1 potentiate FSH action [12]. Thus, during periods of substantial body growth, endocrine Igf1 may increase FSH signaling, which in turn promotes ovarian growth. In support of this idea, pituitary FSH consistently correlated with plasma E2 and Igf1 levels, and strong correlations with plasma FSH and plasma E2 levels were observed in the cortical alveolus stage.

In summary, effects of body growth on previtellogenic oocyte growth in coho salmon were found. Growth during the fall and spring months was strongly related to the degree of oocyte development reached by the spring, with larger fish possessing more advanced oocytes than smaller, slower-growing fish. The accumulation of cortical alveoli was associated with increases in FSH, E2, and ovarian star gene expression, and correlation analyses suggested that the regulation of these factors were related. There was little evidence to suggest that the intraovarian GH-IGF system played a major role in this stage of development. As oocytes accumulated lipid droplets a general increase of components of the FSH-ovarian steroid system and intraovarian igfs occurred. A consistent positive relationship among levels of plasma Igf1 and E2 and pituitary FSH during spring growth suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development. However, the exact nature of the regulatory interactions among these hormones remains unclear, and it is not known whether plasma Igf1 has a significant influence on oocyte growth during the fall and winter months. To further understand the complex mechanisms controlling oocyte growth, future studies will be directed toward localization of the ovarian gene transcripts measured in this study and will use an in vitro approach to examine the possible interplay among FSH, E2, Gh, Sl, and Igfs during previtellogenic growth.

ACKNOWLEDGMENTS

We thank William Fairgrieve, Kathy Cooper, and Nicholas Hodges (Northwest Fisheries Science Center) for assistance with fish maintenance, immunoassays, and histology, respectively. We also thank Dr. Walt Dickhoff (Northwest Fisheries Science Center) for his critical comments during the preparation of the manuscript.

FOOTNOTES

1 Supported by a contract from the Bonneville Power Administration (93–056 to P.S.) and the National Marine Fisheries Service. Presented in part at the 7th International Symposium on the Reproductive Physiology of Fish, Japan (18–23 May 2003). Back

2 Correspondence: Penny Swanson, Northwest Fisheries Science Center, NOAA-National Marine Fisheries Service, 2725 Montlake Blvd. East, Seattle, WA 98112. FAX: 206 860 3467; penny.swanson{at}noaa.gov Back

3 Current address: Hawaii Institute for Marine Biology, P.O. Box 1346 (46–007 Lilipuna Rd.), Kaneohe, HI 96744. Back

4 Current address: Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan. Back

Received: 25 November 2005.

First decision: 20 December 2005.

Accepted: 22 March 2006.

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