General and Comparative Endocrinology xxx (2015) xxx–xxx
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Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria) José M. Guzmán a,⇑, J. Adam Luckenbach a,b, Denis A.M. da Silva a, Gina M. Ylitalo a, Penny Swanson a,b a Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration – National Marine Fisheries Service, Seattle, WA 98112, USA b Center for Reproductive Biology, Washington State University, Pullman, WA 99164, USA
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Article history: Available online xxxx Keywords: Dopamine Puberty Gonadotropins GnRHa DHT Androgens
a b s t r a c t Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17a-hydroxyprogesterone, androstenedione (A4), T and 17b-estradiol (E2) production. Treatment with T increased production of A4, 11b-hydroxyandrostenedione, 11b-hydroxytestosterone, E2, 11-ketotestosterone, and 5a-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Puberty in fishes is the developmental period during which an individual becomes capable of sexually reproducing for the first time, and implies a functional competence of the brain–pituitary–gonad (BPG) axis (Okuzawa, 2002). In females, the onset of puberty is characterized by the first batch of oocytes beginning to actively accumulate cortical alveoli, prior to vitellogenesis, and culminates with the first ovulation (Taranger et al., 2010). Control of reproduction is necessary for the sustainability of commercial aquaculture production; however, while important ⇑ Corresponding author. Fax: +1 206 860 3467. E-mail address: [email protected] (J.M. Guzmán).
advances have been made in the induction of final oocyte maturation, ovulation and spermiation in captive fishes (Mylonas et al., 2010), few advances have been made related to induction and control of puberty. Although the precise mechanisms that trigger the activation of the BPG axis are still unknown, the onset of puberty in fishes is generally marked by activation of the brain gonadotropin-releasing hormone (Gnrh) neuronal system, which regulates the synthesis and release of pituitary gonadotropins (i.e., follicle-stimulating hormone; Fsh, and luteinizing hormone; Lh) (Carrillo et al., 2009; Ojeda et al., 2006; Zohar et al., 2010). Gonadotropins are released into the bloodstream to act in the gonad, stimulating the synthesis of sex steroid hormones (androgens, estrogens and progestogens) and ultimately, the onset of the ovarian growth (Lubzens et al.,
http://dx.doi.org/10.1016/j.ygcen.2015.02.024 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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2010; Taranger et al., 2010). Sex steroids also exert feedback effects on the neuroendocrine reproductive axis, and regulate gonadotropin signaling both directly at the pituitary level and/or indirectly via the brain through the Gnrh system (Quérat et al., 1991; Zanuy et al., 1999). In some species, positive actions of the Gnrh system on pituitary gonadotropin signaling are opposed by an inhibitory effect of dopamine, acting through the dopamine D2 receptor (Drd2) (Dufour et al., 2010) and thus affecting the timing of puberty and early ovarian development (Aizen et al., 2005; Vidal et al., 2004). With the exception of freshwater eels (genus Anguilla), most fishes are able to initiate vitellogenesis in captivity (Zohar and Mylonas, 2001). In some species, however, this process may take many years (e.g., grouper, tuna or sturgeon), increasing costs and risks for aquaculture operations because putative broodstock must be maintained for a prolonged period of time to obtain fertilizable eggs. In these cases, the development of a protocol to advance the age of puberty is needed (Okuzawa, 2002). Hormone therapies are widely used to control reproduction in cultured fish species. However, while generic protocols are usually effective in stimulating ovarian maturation and ovulation in captive species that have fully grown gonads (Guzmán et al., 2009; Mylonas et al., 2007, 2010), those that fail to initiate vitellogenesis are relatively rare and often require species-specific considerations with regard to how the reproductive system is endogenously regulated. For example, in the European eel (Anguilla anguilla) dopamine has been implicated in the blockade of puberty at the silver stage (Dufour et al., 1988; Sébert et al., 2008). For this species, removal of the dopamine inhibition using a dopamine-receptor antagonist (pimozide) in combination with gonadotropin-releasing hormone agonist (GnRHa) and testosterone (T) was required to stimulate pituitary Lh synthesis and release, and initiate vitellogenesis (Vidal et al., 2004). In contrast, a single treatment with GnRHa was sufficient to increase pituitary transcripts for fshb and lhb and serum levels of Lh, and induce vitellogenesis in 16month old juvenile red seabream (Pagrus major) (Kumakura et al., 2003). Interestingly, although a similar treatment also increased serum levels of Lh in 12-month old red seabream, vitellogenesis was not initiated, suggesting that pituitaries of the younger fish responded to the treatment but the ovary was not yet reproductively competent (Okuzawa, 2002). In the Japanese eel (Anguilla japonica), which exhibit severe inhibition of brain– pituitary axis signaling in captivity, only gonadotropin preparations (i.e., pituitary extracts, purified or recombinant gonadotropins) are effective at inducing and promoting vitellogenesis (Sato et al., 2003; Yamauchi and Yamamori, 1982). These treatments act directly on the ovary, and thus their effects are not dependent on activation of pituitary gonadotropin synthesis and secretion. Furthermore, in vitro studies demonstrated that treatments with androgens (T and 11-ketotestosterone; 11KT) and 17b-estradiol (E2) stimulate ovarian follicle growth and accumulation of cortical alveoli, respectively, in juvenile fishes (Forsgren and Young, 2012; Kortner et al., 2009; Lokman et al., 2007), suggesting that sex steroids play a direct role in the ovary during the stage prior to the active accumulation of vitellogenin. Sablefish (Anoplopoma fimbria) is a groundfish native to the North Pacific Ocean ranging from Baja California to Alaska’s Bering Sea and Japan (McFarlane and Beamish, 1983). Due to its rapid growth rate and high market value, sablefish has been identified as an excellent marine aquaculture species. However, efforts to establish sustainable and efficient production have been constrained by the reproductive performance of females from the first-filial generation (F1, produced and maintained in captivity). Although some cultured females may mature at 5+ years old in the industry setting, some other females do not mature during the first 10 years of age and it is not known when, if ever, they
would initiate puberty in captivity. In contrast, F1 cultured males mature at age 2+. This is particularly a problem for selective breeding for high growth because females grow faster and larger than males (Mason et al., 1983), so selection through the female germline may be very important. There is little information on the reproductive physiology of sablefish. A protracted juvenile stage has been reported in the wild as about fifty percent of females first mature at 5 years of age and 58 cm in length (Mason et al., 1983). In a previous study we demonstrated that wild-maturing female sablefish had higher levels of pituitary gonadotropin subunit and ovarian gonadotropin receptor mRNAs and plasma sex steroids compared to 8-year-old, F1 females, which were holding at the immature, perinucleolus stage (Guzmán et al., 2013). These findings suggested that culture conditions might have a suppressing effect on the pituitary gonadotropin–ovary axis of female sablefish, and ultimately block the onset of puberty. Recently, we found drd2 transcripts in the ovary of juvenile sablefish through 454-pyrosequencing (Luckenbach et al., unpublished), suggesting that dopamine may have a regulatory role in the reproduction of this species. Here we describe a series of studies aimed at improving our understanding of the regulation of the reproductive axis in prepubertal female sablefish, which would ultimately lead to development of methods to control the age of puberty. These experiments aimed to: (1) investigate the existence of a dopamine inhibitory tone on the reproductive axis of sablefish; (2) characterize the effect of different doses of T on the pituitary–ovary axis of prepubertal female sablefish; (3) determine whether combined treatments with T, GnRHa and a dopamine D2 receptor antagonist (metoclopramide, Met) can stimulate the reproductive axis and potentially be used to induce puberty in female sablefish; and (4) evaluate the steroidogenic capability of ovaries of prepubertal sablefish exposed in vitro to T, Met and gonadotropin preparations, by using liquid chromatography/tandem mass spectrometry (LC– MS/MS).
2. Materials and methods 2.1. Experimental animals and treatment protocols 2.1.1. Study 1: tissue distribution of sablefish drd2 transcripts and comparison of expression levels in brain, pituitary and ovary between prepubertal and maturing females Partial cDNA sequences encoding sablefish drd2 were obtained by 454-pyrosequencing of differentiating ovaries of juvenile sablefish (Luckenbach et al., unpublished). Complementary DNA reads were assembled using Newbler Assembler version 2.6 bioinformatics software (Roche, Indianapolis, IN), and a partial cDNA sequence for drd2 was confirmed by BLASTN (National Center for Biotechnology Information, NCBI). Tissue distribution analysis of drd2 was conducted using semiquantitative reverse transcriptase polymerase chain reaction (semi-qPCR). Total RNA was isolated from 10 different tissues (brain, pituitary, ovary, testis, heart, gills, kidney, intestine, spleen and liver) of maturing wild-caught sablefish from a previous study (Guzmán et al., 2013). RNA was isolated, treated with DNase and reverse-transcribed as detailed in Section 2.2. The semi-qPCRs for drd2 and elongation factor 1 alpha (eef1a, assessed to verify quality and equal loading of cDNA) had a total volume of 25 ll and consisted of 1 GoTaq Green Master Mix (Promega), 0.4 lM of the forward and reverse primer (Supplementary Table 1) and 100 ng of cDNA template based on the amount of total RNA loaded into the reverse transcription (RT) reactions. Thermal cycling was performed on an iCycler Thermocycler (BioRad) using an initial denaturation at 94 °C for 2 min followed by 40 or 32 cycles (for drd2 and
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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eef1a, respectively) of 94 °C for 30 s (denaturation), 59 °C for 30 s (annealing) and 72 °C for 30 s (elongation), and a final elongation of 72 °C for 7 min. Products were electrophoresed on 2% agarose E-Gels (Life Technologies, Carlsbad, CA). Differences in the expression of pituitary and ovary drd2 between prepubertal and maturing females were determined by qPCR. For this, we used cDNAs previously employed to characterize gonadotropin subunit and receptor gene expression in sablefish (Guzmán et al., 2013), while brain cDNA was obtained as described in Section 2.2. In this study, wild sablefish were caught near the mouth of the Quinault River, off the coast of Washington in October 2010 and transported to the Northwest Fisheries Science Center (NWFSC), Manchester Marine Research Station (Port Orchard, WA). During February 2011, wild females showed signs of sexual maturation by ultrasound. In parallel, a group of F1 female sablefish (broodyear 2003) produced at the Manchester Marine Research Station was transported to the NWFSC hatchery facilities in Seattle (WA) in fall 2010. The fish were maintained under similar rearing conditions as wild-caught fish, but had never undergone puberty. Both wild maturing and prepubertal F1 females were sampled during the spawning season of 2011 for tissue collection (see details in Guzmán et al., 2013). 2.1.2. Study 2: effect of different doses of T on the pituitary–ovary axis of prepubertal females F1 sablefish (broodyear 2011) were produced at the Manchester Marine Research Station and transported to the NWFSC hatchery facilities in Seattle in January 2012. Female and male fish were maintained together in 4 m3 indoor tanks supplied with recirculated seawater (12.1 ± 0.1 °C), and exposed to artificial lighting with simulated natural photoperiod. Fish were fed ad libitum on dry pellets (BioBrood, Bio-Oregon, Longview, WA). In January 2013, four females were sampled to determine the initial reproductive stage (pre-treatment, time 0). Fish were anesthetized by immersion in tricaine methanesulfonate (MS-222, Argent, Redmond, WA) and body weight (BW) and fork length (FL) recorded (1187.4 ± 49.4 g BW, 46.9 ± 0.5 cm FL). Blood (1 ml) was collected from the caudal vasculature with heparinized syringes and placed in glass tubes on crushed ice. Plasma was obtained by centrifugation of whole blood (3000g, 15 min, 4 °C) and stored at 20 °C for sex steroid (T, E2 and 11KT) analyses by enzyme-linked immunosorbent assays (ELISAs). Anesthetized fish were euthanized by decapitation for the collection of tissues. Pituitary glands were collected, immediately frozen in liquid nitrogen and stored at 80 °C until total RNA extraction for gonadotropin subunits (fshb, lhb and gpa) and drd2 gene expression analyses by qPCR. Ovaries and liver were removed and weighed for calculation of the gonadosomatic index (GSI = gonad weight 100/BW) and hepatosomatic index (HSI = liver weight 100/BW). For histology, a middle portion of the ovary was preserved in Bouin’s fixative for 24 h before storage in 70% ethanol. Subsequently, 17 females from the same cohort (1252.4 ± 44.3 g BW, 47.7 ± 0.5 cm FL) were randomly distributed into four groups (n = 4–5 fish/group) and implanted intramuscularly with sustained-release cholesterol pellets (30 mg, 95% cholesterol and 5% cellulose) containing T at doses of 0 (control), 0.15, 0.75 or 3.75 mg. Blood (0.7 ml) was collected on days 3, 7, 14, 21 and 28 after being implanted and the time course of sex steroids was determined. On day 28, all fish were euthanized as described above, and tissues (blood, pituitary, ovary and liver) were collected to examine the effect of different doses of T on the pituitary–ovary axis. Males in the tank were also treated with T and sampled as described for females, but results are not included in this study. Before study 2 was initiated, the release kinetics from the sustained-release cholesterol pellets containing T was examined in vitro. Briefly, implants containing 0, 0.15, 0.75 and 3.75 mg of
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T (n = 4 implants/dose) were placed in a 48-well plate (one implant/well) containing 500 ll of in vitro buffer (ELISA buffer, 0.1 M potassium buffer, pH 7.4). The plate was placed on a rocking shaker in a controlled-temperature incubator at 12 °C. On day 1, 3, 7, 14, 21 and 28, the incubation buffer was taken from each well and stored at 30 °C until analysis of T by ELISA. The buffer was refreshed after each sampling. In vitro release profiles are shown in Supplementary Fig. 1. 2.1.3. Study 3: effect of long-term T, GnRHa and the dopamine D2receptor antagonist, Met, treatments on the pituitary–ovary axis of prepubertal female sablefish F1 sablefish (broodyear 2010) were produced at the Manchester Research Station and held in net-pens in Clam Bay until winter 2013. Water temperature in the net-pens fluctuated seasonally, reaching minimum values in February and maximum in August (average temperature was 10.9 ± 0.2 °C; 7.7 °C min. and 14.9 °C max. corresponding to February and August 2012, respectively). In January 2013, fish were transported to three different outdoor tanks (4 m3) supplied with flow-through, sand-filtered and UVtreated seawater. They had been previously implanted with passive-integrated transponder tags and a blood sample taken so that sex could be genetically determined using genomic DNA from red blood cells (Rondeau et al., 2013). Only females were selected for this study, and exposed to natural photoperiod and water temperature. During the experiment, the water temperature increased naturally from 10.4 to 13.5 °C (average temperature for this period was 12.7 ± 0.2 °C) from 15 May to 18 July. Fish were fed ad libitum on dry pellets. In May 2013 (coinciding with the onset of the ovarian vitellogenic growth in the wild, Guzmán et al., unpublished), six 3-year old F1 female sablefish were sampled to determine the initial stage (pre-treatment, time 0). BW and FL were recorded (3411.3 ± 193.8 g BW, 62.7 ± 1.3 cm FL) and tissues (blood, pituitary, ovary and liver) were collected. On the same day, 48 females from the same cohort were anesthetized, BW and FL recorded (3649.6 ± 54.2 g BW, 64.8 ± 0.3 cm FL) and implanted every 3 weeks over 9 weeks with intramuscular, sustained-release cholesterol pellets containing: blank (control), T (10 mg), GnRHa ([des-Gly10, D-Ala6]-LH-RH, 200 lg) and Met (10 mg) alone or in combination. Every 3 weeks (coinciding with the next implantation), blood (1 ml) was collected from all fish to track levels of sex steroids. Three weeks after receiving the last implant, all fish were euthanized and tissues collected to examine the effect of T, GnRHa and Met, alone or in combination. 2.1.4. Study 4: steroidogenic capability of ovaries of prepubertal sablefish in vitro The same group of F1 female sablefish (broodyear 2003) that was used for study 1, and previously employed to characterize the reproductive endocrine axis in this species (Guzmán et al., 2013), was used for study 4. In August 2012, five 9 year-old females (3190 ± 124 g BW, 64.3 ± 8.9 cm FL) were sampled to obtain ovaries for in vitro culture. The ovaries were cut into 40 mg pieces and distributed into 24-well polystyrene culture plates so that each well received one piece of the ovaries from an individual fish. Culture wells contained 1 ml of Cortland’s solution supplemented with 0.2% BSA. An ovarian subsample was also collected for histology. Tissues were pre-incubated at 12 °C with gentle orbital shaking at 100 rpm for 1 h. After the pre-incubation, the medium was removed and replaced with either fresh medium alone (control) or medium containing T (1, 10 and 100 ng/ml), Met (10, 100 and 1000 lM), GnRHa (10, 100, 1000 nM), human chorionic gonadotropin (hCG, 0.1, 1 and 10 IU/ml) or pregnant-mare serum gonadotropin (PMSG, 0.1, 1 and 10 IU/ml). The highest doses of T
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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(100 ng/ml), Met (1 mM) and hCG (10 IU/ml) were also tested in combination. GnRHa and PMSG were not included in the combination because in previous ovarian incubations we observed no steroidogenic effect of GnRHa, while the effect of PMSG was similar to hCG. After a 24-h incubation, the culture medium was collected and stored at 80 °C until sex steroid analysis by LC–MS/MS. 2.2. RNA isolation and cDNA synthesis Total RNA from sablefish tissues was isolated with Tri-Reagent (Molecular Research Center, Cincinnati, OH) using a TissueLyser II (Qiagen, Valencia, CA). For partial cDNA cloning and gene expression analysis, a total RNA subsample was diluted to 250 ng RNA/ll in nuclease-free water and DNase treated using the Turbo DNA Free kit’s protocol (Ambion, Life Technologies, Grand Island, NY). RNA yields and quality were assessed by NanoDrop (ND1000 Spectrophotometer; NanoDrop Technologies, Rockland, DE) and gel electrophoresis. For RT, 500 ng of total RNA of each sample was reverse transcribed in 10-ll reactions with the Superscript II kit (Invitrogen, Life Technologies). Other necessary components for RT, such as random primers and RNase inhibitor, were purchased from Promega (Madison, WI). Negative control reactions were performed without the addition of the RT enzyme for a subset of the RNA samples. 2.3. Quantitative real-time PCR Quantitative PCR assays were run on an ABI 7900HT Fast RealTime PCR System (Life Technologies) in 384-well plates using standard cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Reactions were 12.5 ll each and consisted of 1 Power SYBR Green master mix (Applied Biosystems, Life Technologies), 150 nM of the forward and reverse primer (Supplementary Table 1), and 0.5 ng cDNA template based on the amount of total RNA loaded into the RT reactions. Standard curves were generated from serial dilution of cDNA pooled from RNA samples obtained from the target tissue (i.e., brain, pituitary, or ovary; one RNA sample representative of each group or treatment was included in the standard). Standard curve samples ranged from 0.1 to 5 ng cDNA, based on the amount of RNA added to the RT reactions, and represented 4 dilutions over this range. Linearity of the standard curve was confirmed for the assays and results were analyzed using the relative standard curve method. Each standard curve cDNA dilution was run in triplicate, while each sample was run in duplicate. For each gene, within a study, all samples were assayed in the same plate to avoid across plate variation. A dissociation or melt curve analysis step was included in each qPCR to confirm the assay specificity. Negative controls included in each plate consisted of either no cDNA template or RNA that was not reverse transcribed. These controls showed no detectable amplification over 40 cycles of PCR. Results for each target gene were normalized to eef1a which was demonstrated to be stable across groups in all tissues (results not shown). To improve the presentation of results, the mean value of the wild maturing (study 1) or time-0 fish (study 2 and 3) was set to 1. 2.4. Sex steroid analyses 2.4.1. Steroid analysis by ELISA In the in vivo studies (study 2 and 3), as well to determine the in vitro release of T loaded in the cholesterol pellets (study 2), levels of sex steroids were quantified by ELISA using protocols validated for sablefish (Guzmán et al., 2013). The sensitivities of the assays, calculated as maximum binding minus twice the standard
deviation, were 11.9, 2.8 and 4.9 pg/ml for the E2, T and 11KT immunoassays, respectively. The ED50s for the E2, T, and 11KT assays were 1.32, 0.15 and 0.07 ng/ml, respectively. Intra-assay coefficients of variation (CVs; n = 10) were 5.7%, 4.1% and 3.7% for E2, T and 11KT, respectively. Inter-assay CVs were 7.3%, 13.7% and 11.2% for E2, T and 11KT, respectively. Other details on cross-reactivity of E2, T and 11KT antibodies, and correlation between levels of E2, T and 11KT measured by ELISA and LC–MS/ MS in plasma samples of sablefish are provided in Supplementary Figs. 2–4. 2.4.2. Steroid analysis by LC–MS/MS Levels of sex steroids in ovarian incubation media (study 4) were determined by LC–MS/MS using a modified method described previously (Koren et al., 2012). Samples were extracted and cleaned using solid-phase extraction. A 250 ll aliquot of culture medium was diluted with water (1:1, v/v), followed by the addition of 20 ll of a mixture of surrogate standards (S-std) containing 11b-hydroxyandrostendione-d7, 17b-estradiol-d4 and progesterone-d9 in methanol (250 ng/ml), 100 ll of glacial acetic acid and 1 ml of water. The solution was mixed and loaded into a 3 ml Strata-X reversed-phase cartridge containing 60 mg of sorbent (Phenomenex Inc, Torrance, CA, USA) previously conditioned with 2 ml of methanol, followed by the addition of 2 ml of water. After the sample was loaded, the cartridge was washed with a mixture of methanol/water (60/40, v/v). The cartridge was dried under vacuum for 30 min and the target steroids were eluted with 1.5 ml of methanol into clean 2-ml vials. The volume of the final extract was reduced to 0.5 ml under a stream of nitrogen gas at room temperature. Estrone-d4 was added as an internal standard (IS; 20 ll of a 250 ng/ml methanolic solution) and an aliquot was transferred into a vial for injection into the LC– MS/MS. The sample extracts were analyzed by LC (Acquity system, Waters Co., Milford, MA) coupled with a triple quadrupole tandem mass spectrometer (MS/MS, QTRAP 5500, AB Sciex, Framingham, MA). For each sample, 10 ll of extract was injected onto the LC that was equipped with a 0.2-lm pre-filter followed by a 2.1 5.0 mm (1.7-lm particle size) C18 guard column and a 2.1 150 mm (1.7 lm particle size) reversed-phase column. Water (solvent A) and methanol (solvent B) were used as the mobile-phase. The total analysis time was 30 min using a linear gradient, as follows (solvent A/solvent B): initial gradient was 60/40 at 0.2 ml/min; 14 min to 36/64 at 0.2 ml/min; 5 min to 20/80 at 0.2 ml/min; 0.1 min to 100% solvent B at 0.2 ml/min; 0.1 min to increase the flow up to 0.35 ml/min and held for 4.8 min; 0.1 min to reduce flow to 0.30 ml/min; 0.9 min to initial gradient 60/40 at 0.3 ml/ min and held for 5 min. The column temperature was maintained at 45 °C. Electrospray ionization (ESI) mode was used for the ionization of all analytes. The MS/MS was operating in both negative and positive ion mode and the steroids were detected via multiple-reaction monitoring (MRM). The ion source was kept at 700 °C and capillary voltage varied between 4.5 kV (negative mode) and 5.5 kV (positive mode). Other details on the MRM parameters are given in the Supplementary Table 2. The analytes were quantified by S-std and based on the calibration curve of each analyte. The recovery of each S-std was calculated using the internal standard. The LC–MS/MS method used to measure steroids was validated by spiking the analytes at 0.1 ng/ml into culture medium before and after the extraction/cleanup on the solid-phase extraction. Using this approach, the recovery of steroids and any potential matrix effect were evaluated. The percent recovery for all analytes in both tests varied from 70% to 120%. Spiking solutions contained all of the target analytes as well as deuterated surrogate and internal standards used for quantifying the steroids
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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and calculating the recovery of the surrogate standards. A method blank (LC–MS grade water), a spiked blank and a spiked matrix sample were analyzed with each sample batch. The percent recoveries of the steroids in the spiked samples ranged from 81% to 116%. The recoveries of the surrogate standards in both culture medium samples and associated quality assurance samples ranged from 67% to 107%. The LC–MS/MS system was calibrated using 5 different levels of steroids ranging from 0.03 to 30 ng/ml (in methanol) and surrogate standards were used to calculate the analyte concentrations. Another methanolic standard solution (LC-QC) was injected multiple times before, during and after each sequence of sample extracts in order to measure the stability of the instrument response. The relative standard deviation for the LC-QC injections within each sequence was less than 15% for all analytes. Limit of quantitation was compound dependent and varied from 0.02 to 0.6 ng/ml.
2.5. Histology and image analysis Fixed ovaries were dehydrated through a graded series of ethanol and embedded in paraffin wax, sectioned at a thickness of 4 lm, and stained with hematoxylin and eosin. Microscope images were captured using a Nikon (Melville, NY) video camera and analyzed through NIS Elements image software version 2.3 (Nikon). For analysis, the 30 largest follicles contained within a frame of 3.5 mm2 were selected and their areas measured by NIS Elements image software. Thirty follicles were measured in three non-overlapping frames per section. The radius (r) was calculated from the circumference and used to calculate follicle volume (V) according to the formula: V = 4/3pr3. We defined the maximum ovarian follicle volume per female as the average volume of the largest follicles (n = 90).
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2.6. Statistical analyses Statistical analyses were performed using Prism 5 software for Mac OSX (GraphPad Software, La Jolla, CA) with the minimum level of significance set to P < 0.05. In study 1, differences between prepubertal F1 and maturing females were assessed with a Student’s t-test. In study 2 and 3, differences in the in vitro release of T loaded in the cholesterol pellets, and in plasma sex steroid levels were analyzed using a two-way ANOVA with dose/treatment and time as independent variables and where significant differences were observed, Bonferroni post-tests were conducted. All other parameters analyzed in study 2–4 were assessed by oneway ANOVA and where significant differences were observed, Tukey multiple comparisons tests were conducted. For statistical purposes, in study 4, the lowest detectable value was assigned to samples below the limit of quantitation. When necessary to normalize distribution, values were Log-transformed prior to the ANOVA. Data are expressed as mean ± standard error of the mean (SEM).
3. Results 3.1. Study 1: tissue distribution of sablefish drd2 transcripts and comparison of expression levels in brain, pituitary and ovary between prepubertal and maturing females Transcripts for drd2 were found in all the reproductive tissues examined in sablefish, including brain, pituitary, ovary and testis, as well as in heart and spleen (Fig. 1). Levels of drd2 transcripts were highest in the brain, ovary and testis. Both brain and pituitary transcript levels of drd2 were higher in prepubertal F1 females relative to wild maturing females (4- and
Fig. 1. Tissue distribution of dopamine receptor D2 mRNA (drd2) in sablefish (upper) and its relative expression in brain, pituitary and ovary obtained from wild maturing (filled columns) and prepubertal F1 female sablefish (open columns) (lower). Transcript levels of eef1a were assessed to verify quality and equal loading of cDNAs. Gene expression levels were determined by qPCR and normalized to eef1a. Data are expressed as the mean ± SEM (n = 3). Columns with an asterisk are significantly different (t-test, p < 0.05).
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 2. Effect of different doses of testosterone on the pituitary–ovary axis in F1 female sablefish. Females (1.25 ± 0.04 kg) were treated with a single implant containing 0.15, 0.75 or 3.75 mg of T for 28 days. (A) Plasma profile of sex steroid levels, determined by ELISA. (B) Pituitary gene expression levels of gonadotropin subunits and (C) dopamineD2 receptors, determined by qPCR and normalized to eef1a. (D) Representative micrographs of ovaries from control (1) or females treated with 0.75 mg of T (2) after 28 days of treatment. (E) Maximum ovarian follicle volume, calculated by measuring the maximum area of the most advanced follicles (n = 90) per female using NIS-Elements Version 2.3. Data are expressed as the mean ± SEM (n = 4–5). Values with different superscript letters are significantly different, whereas asterisks (⁄) indicate significant differences from time 0 (ANOVA, p < 0.05).
30-fold respectively), whereas no significant differences were observed for drd2 transcripts in the ovary (Fig. 1). 3.2. Study 2: effect of different doses of T on the pituitary–ovary axis of prepubertal females The effects of sustained-release cholesterol pellets containing T at doses of 0.15, 0.75 and 3.75 mg on plasma levels of sex steroids (T, E2 and 11KT) in prepubertal F1 female sablefish for 28 days are shown in Fig. 2A. The low dose of T (0.15 mg) did not affect plasma levels of T or E2 but increased plasma 11KT levels at day 7 and 14. The mid dose of T (0.75 mg) increased levels of plasma T for 14 days and 11KT at days 7, 14 and 21, but did not increase E2 levels relative to control. In contrast, the high dose of T (3.75 mg) induced significant increases in plasma T levels for 28 days, and both E2 and 11KT for 21 days. At the pituitary level, sustained administration of T did not significantly affect transcript levels for fshb or gpa relative to control, although the low dose of T (0.75 mg) increased transcripts for fshb relative to time 0 (Fig. 2B). The highest dose of T (3.75 mg) induced a significant increase in lhb transcripts (elevated 35-fold relative to
control fish). Treatments with T did not affect pituitary drd2 transcripts (Fig. 2C). As expected, the ovaries of females at the start of the study (time 0, data not shown) were characterized by the presence of follicles in primary growth (perinucleolus stage). Treatments with T did not stimulate the initiation of secondary oocyte growth (i.e., the appearance of cortical alveoli or yolk granules), and ovarian follicles remained at the perinucleolus stage (Fig. 2D). However, treatments with mid and high doses of T induced a significant increase in the maximum volume of ovarian follicles after 28 days of treatment (Fig. 2E). No effect of treatments on GSI or HSI was observed (Supplementary Fig. 5). 3.3. Study 3: effect of long-term T, GnRHa and the dopamine D2receptor antagonist, Met, treatments on the pituitary–ovary axis of prepubertal female sablefish The effects of sustained-release cholesterol pellets containing T (10 mg), GnRHa (200 lg) and Met (10 mg) alone or in combination on plasma levels of sex steroids (T, E2 and 11KT) in prepubertal F1 female sablefish for 9 weeks are shown in Fig. 3A. Only T-
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 3. Effect of testosterone (T), metoclopramide (Met) and gonadotropin-releasing hormone agonist (GnRHa) on the pituitary–ovary axis in F1 female sablefish. Fish (3.65 ± 0.05 kg) were treated every 3 weeks with implants containing T (10 mg), Met (10 mg) or GnRHa (200 lg) alone or combined, for 9 weeks. (A) Plasma profile of sex steroid levels, determined by ELISA. (B) Pituitary gene expression levels of gonadotropin subunits and (C) dopamine-D2 receptors, determined by qPCR and normalized to eef1a. (D) Representative micrographs of ovaries from control females (1) or those treated with T (2), Met (3) or GnRHa (4) after 9 weeks. (E) Ovarian follicle volume, calculated by measuring the maximum area of the most advanced follicles (n = 90) per female using NIS-Elements software. Data are expressed as the mean ± SEM (n = 6). Values with different superscript letters are significantly different, whereas asterisks (⁄) indicate significant differences from time 0 (ANOVA, p < 0.05).
containing implants increased plasma levels of sex steroids. Specifically, T and 11KT were maintained at elevated levels over the 9 week experiment, while levels of E2 were elevated in females treated with T alone or combined with Met and GnRHa at week 3, and in those treated with T combined with Met and/or GnRHa at week 9. At the pituitary level, sustained administration of Met (alone) increased the expression of fshb, but had no effect on lhb or gpa transcripts (Fig. 3B). When Met was combined with T and/or GnRHa the fshb transcripts were not significantly different than controls. Both T and GnRHa significantly affected the transcript levels of lhb (70- and 7-fold respectively), and the combination of T and GnRHa had a strong synergistic effect on the expression of lhb (2000-fold higher than control). This combination of T and GnRHa was also effective in elevating transcripts of gpa, and the effect was enhanced when Met was included. Conversely, pituitary drd2 transcripts were not affected by any of the treatments (Fig. 3C). The ovaries of females were also characterized by the presence of follicles in primary growth (perinucleolus stage) at time 0 (results not shown). None of the treatments tested induced the
transition to secondary oocyte growth (Fig. 3D). However, as also observed in study 2, administration of T was effective in elevating the maximum ovarian follicle volume relative to controls (Fig. 3E). A slight but significant effect on the maximum ovarian follicle volume was also observed in females treated with Met (Fig. 3E), although the maximum ovarian follicle volume in controls was lower than that of fish sampled at time 0 and Met did not significantly increase follicle volume over fish at time 0. No significant effects of treatments on GSI or HSI were observed (Supplementary Fig. 6). 3.4. Study 4: steroidogenic capability of primary follicles in vitro Effects of T, Met and hCG alone and in combination on the in vitro ovarian production of various sex steroids is shown in Fig. 4. From a total of 15 steroids simultaneously analyzed by LC–MS/MS (see complete list in Supplementary Table 2), only 8 of them were detectable in the culture medium. These included 17a-hydroxyprogesterone (17OHP4), androstenedione (A4), 11bhydroxyandrostenedione (11OHA4), 11b-hydroxytestosterone (11OHT), T, E2, 5a-dihydrotestosterone (DHT) and 11KT.
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 4. In vitro production of sex steroids by perinucleolus stage ovarian fragments of F1 sablefish exposed to testosterone (100 ng/ml), metoclopramide (1 mM) and human chorionic gonadotropin (hCG, 10 IU/ml) alone or combined. Ovarian fragments (40 mg) were cultured in Cortland’s solution for 24 h at 12 °C. Data are expressed as the mean ± SEM (n = 5). Values with different superscript letters are significantly different (ANOVA, p < 0.05). For statistical purposes, the lowest detectable value was assigned to samples below the detection limit (indicated by a dotted line). In these cases, asterisks (⁄) indicate significant differences from the control group (ANOVA, p < 0.05).
As expected, the ovaries used for this study were also at the perinucleolus stage. Only E2 was detected in the culture medium of control samples (ranged from 0.06 to 0.13 ng/ml). In general, T was the most potent treatment in increasing the production of A4, 11OHA4, 11OHT, E2, DHT and 11KT (Fig. 4 and Supplementary Fig. 7). Ovarian fragments incubated with the highest dose of T (100 ng/ml) preferentially produced A4 (>10 ng/ml) and DHT (>5 ng/ml) over other metabolites (11OHA4, 11OHT, E2, 11KT,
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Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria) José M. Guzmán a,⇑, J. Adam Luckenbach a,b, Denis A.M. da Silva a, Gina M. Ylitalo a, Penny Swanson a,b a Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration – National Marine Fisheries Service, Seattle, WA 98112, USA b Center for Reproductive Biology, Washington State University, Pullman, WA 99164, USA
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Article history: Available online xxxx Keywords: Dopamine Puberty Gonadotropins GnRHa DHT Androgens
a b s t r a c t Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17a-hydroxyprogesterone, androstenedione (A4), T and 17b-estradiol (E2) production. Treatment with T increased production of A4, 11b-hydroxyandrostenedione, 11b-hydroxytestosterone, E2, 11-ketotestosterone, and 5a-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Puberty in fishes is the developmental period during which an individual becomes capable of sexually reproducing for the first time, and implies a functional competence of the brain–pituitary–gonad (BPG) axis (Okuzawa, 2002). In females, the onset of puberty is characterized by the first batch of oocytes beginning to actively accumulate cortical alveoli, prior to vitellogenesis, and culminates with the first ovulation (Taranger et al., 2010). Control of reproduction is necessary for the sustainability of commercial aquaculture production; however, while important ⇑ Corresponding author. Fax: +1 206 860 3467. E-mail address: [email protected] (J.M. Guzmán).
advances have been made in the induction of final oocyte maturation, ovulation and spermiation in captive fishes (Mylonas et al., 2010), few advances have been made related to induction and control of puberty. Although the precise mechanisms that trigger the activation of the BPG axis are still unknown, the onset of puberty in fishes is generally marked by activation of the brain gonadotropin-releasing hormone (Gnrh) neuronal system, which regulates the synthesis and release of pituitary gonadotropins (i.e., follicle-stimulating hormone; Fsh, and luteinizing hormone; Lh) (Carrillo et al., 2009; Ojeda et al., 2006; Zohar et al., 2010). Gonadotropins are released into the bloodstream to act in the gonad, stimulating the synthesis of sex steroid hormones (androgens, estrogens and progestogens) and ultimately, the onset of the ovarian growth (Lubzens et al.,
http://dx.doi.org/10.1016/j.ygcen.2015.02.024 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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2010; Taranger et al., 2010). Sex steroids also exert feedback effects on the neuroendocrine reproductive axis, and regulate gonadotropin signaling both directly at the pituitary level and/or indirectly via the brain through the Gnrh system (Quérat et al., 1991; Zanuy et al., 1999). In some species, positive actions of the Gnrh system on pituitary gonadotropin signaling are opposed by an inhibitory effect of dopamine, acting through the dopamine D2 receptor (Drd2) (Dufour et al., 2010) and thus affecting the timing of puberty and early ovarian development (Aizen et al., 2005; Vidal et al., 2004). With the exception of freshwater eels (genus Anguilla), most fishes are able to initiate vitellogenesis in captivity (Zohar and Mylonas, 2001). In some species, however, this process may take many years (e.g., grouper, tuna or sturgeon), increasing costs and risks for aquaculture operations because putative broodstock must be maintained for a prolonged period of time to obtain fertilizable eggs. In these cases, the development of a protocol to advance the age of puberty is needed (Okuzawa, 2002). Hormone therapies are widely used to control reproduction in cultured fish species. However, while generic protocols are usually effective in stimulating ovarian maturation and ovulation in captive species that have fully grown gonads (Guzmán et al., 2009; Mylonas et al., 2007, 2010), those that fail to initiate vitellogenesis are relatively rare and often require species-specific considerations with regard to how the reproductive system is endogenously regulated. For example, in the European eel (Anguilla anguilla) dopamine has been implicated in the blockade of puberty at the silver stage (Dufour et al., 1988; Sébert et al., 2008). For this species, removal of the dopamine inhibition using a dopamine-receptor antagonist (pimozide) in combination with gonadotropin-releasing hormone agonist (GnRHa) and testosterone (T) was required to stimulate pituitary Lh synthesis and release, and initiate vitellogenesis (Vidal et al., 2004). In contrast, a single treatment with GnRHa was sufficient to increase pituitary transcripts for fshb and lhb and serum levels of Lh, and induce vitellogenesis in 16month old juvenile red seabream (Pagrus major) (Kumakura et al., 2003). Interestingly, although a similar treatment also increased serum levels of Lh in 12-month old red seabream, vitellogenesis was not initiated, suggesting that pituitaries of the younger fish responded to the treatment but the ovary was not yet reproductively competent (Okuzawa, 2002). In the Japanese eel (Anguilla japonica), which exhibit severe inhibition of brain– pituitary axis signaling in captivity, only gonadotropin preparations (i.e., pituitary extracts, purified or recombinant gonadotropins) are effective at inducing and promoting vitellogenesis (Sato et al., 2003; Yamauchi and Yamamori, 1982). These treatments act directly on the ovary, and thus their effects are not dependent on activation of pituitary gonadotropin synthesis and secretion. Furthermore, in vitro studies demonstrated that treatments with androgens (T and 11-ketotestosterone; 11KT) and 17b-estradiol (E2) stimulate ovarian follicle growth and accumulation of cortical alveoli, respectively, in juvenile fishes (Forsgren and Young, 2012; Kortner et al., 2009; Lokman et al., 2007), suggesting that sex steroids play a direct role in the ovary during the stage prior to the active accumulation of vitellogenin. Sablefish (Anoplopoma fimbria) is a groundfish native to the North Pacific Ocean ranging from Baja California to Alaska’s Bering Sea and Japan (McFarlane and Beamish, 1983). Due to its rapid growth rate and high market value, sablefish has been identified as an excellent marine aquaculture species. However, efforts to establish sustainable and efficient production have been constrained by the reproductive performance of females from the first-filial generation (F1, produced and maintained in captivity). Although some cultured females may mature at 5+ years old in the industry setting, some other females do not mature during the first 10 years of age and it is not known when, if ever, they
would initiate puberty in captivity. In contrast, F1 cultured males mature at age 2+. This is particularly a problem for selective breeding for high growth because females grow faster and larger than males (Mason et al., 1983), so selection through the female germline may be very important. There is little information on the reproductive physiology of sablefish. A protracted juvenile stage has been reported in the wild as about fifty percent of females first mature at 5 years of age and 58 cm in length (Mason et al., 1983). In a previous study we demonstrated that wild-maturing female sablefish had higher levels of pituitary gonadotropin subunit and ovarian gonadotropin receptor mRNAs and plasma sex steroids compared to 8-year-old, F1 females, which were holding at the immature, perinucleolus stage (Guzmán et al., 2013). These findings suggested that culture conditions might have a suppressing effect on the pituitary gonadotropin–ovary axis of female sablefish, and ultimately block the onset of puberty. Recently, we found drd2 transcripts in the ovary of juvenile sablefish through 454-pyrosequencing (Luckenbach et al., unpublished), suggesting that dopamine may have a regulatory role in the reproduction of this species. Here we describe a series of studies aimed at improving our understanding of the regulation of the reproductive axis in prepubertal female sablefish, which would ultimately lead to development of methods to control the age of puberty. These experiments aimed to: (1) investigate the existence of a dopamine inhibitory tone on the reproductive axis of sablefish; (2) characterize the effect of different doses of T on the pituitary–ovary axis of prepubertal female sablefish; (3) determine whether combined treatments with T, GnRHa and a dopamine D2 receptor antagonist (metoclopramide, Met) can stimulate the reproductive axis and potentially be used to induce puberty in female sablefish; and (4) evaluate the steroidogenic capability of ovaries of prepubertal sablefish exposed in vitro to T, Met and gonadotropin preparations, by using liquid chromatography/tandem mass spectrometry (LC– MS/MS).
2. Materials and methods 2.1. Experimental animals and treatment protocols 2.1.1. Study 1: tissue distribution of sablefish drd2 transcripts and comparison of expression levels in brain, pituitary and ovary between prepubertal and maturing females Partial cDNA sequences encoding sablefish drd2 were obtained by 454-pyrosequencing of differentiating ovaries of juvenile sablefish (Luckenbach et al., unpublished). Complementary DNA reads were assembled using Newbler Assembler version 2.6 bioinformatics software (Roche, Indianapolis, IN), and a partial cDNA sequence for drd2 was confirmed by BLASTN (National Center for Biotechnology Information, NCBI). Tissue distribution analysis of drd2 was conducted using semiquantitative reverse transcriptase polymerase chain reaction (semi-qPCR). Total RNA was isolated from 10 different tissues (brain, pituitary, ovary, testis, heart, gills, kidney, intestine, spleen and liver) of maturing wild-caught sablefish from a previous study (Guzmán et al., 2013). RNA was isolated, treated with DNase and reverse-transcribed as detailed in Section 2.2. The semi-qPCRs for drd2 and elongation factor 1 alpha (eef1a, assessed to verify quality and equal loading of cDNA) had a total volume of 25 ll and consisted of 1 GoTaq Green Master Mix (Promega), 0.4 lM of the forward and reverse primer (Supplementary Table 1) and 100 ng of cDNA template based on the amount of total RNA loaded into the reverse transcription (RT) reactions. Thermal cycling was performed on an iCycler Thermocycler (BioRad) using an initial denaturation at 94 °C for 2 min followed by 40 or 32 cycles (for drd2 and
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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eef1a, respectively) of 94 °C for 30 s (denaturation), 59 °C for 30 s (annealing) and 72 °C for 30 s (elongation), and a final elongation of 72 °C for 7 min. Products were electrophoresed on 2% agarose E-Gels (Life Technologies, Carlsbad, CA). Differences in the expression of pituitary and ovary drd2 between prepubertal and maturing females were determined by qPCR. For this, we used cDNAs previously employed to characterize gonadotropin subunit and receptor gene expression in sablefish (Guzmán et al., 2013), while brain cDNA was obtained as described in Section 2.2. In this study, wild sablefish were caught near the mouth of the Quinault River, off the coast of Washington in October 2010 and transported to the Northwest Fisheries Science Center (NWFSC), Manchester Marine Research Station (Port Orchard, WA). During February 2011, wild females showed signs of sexual maturation by ultrasound. In parallel, a group of F1 female sablefish (broodyear 2003) produced at the Manchester Marine Research Station was transported to the NWFSC hatchery facilities in Seattle (WA) in fall 2010. The fish were maintained under similar rearing conditions as wild-caught fish, but had never undergone puberty. Both wild maturing and prepubertal F1 females were sampled during the spawning season of 2011 for tissue collection (see details in Guzmán et al., 2013). 2.1.2. Study 2: effect of different doses of T on the pituitary–ovary axis of prepubertal females F1 sablefish (broodyear 2011) were produced at the Manchester Marine Research Station and transported to the NWFSC hatchery facilities in Seattle in January 2012. Female and male fish were maintained together in 4 m3 indoor tanks supplied with recirculated seawater (12.1 ± 0.1 °C), and exposed to artificial lighting with simulated natural photoperiod. Fish were fed ad libitum on dry pellets (BioBrood, Bio-Oregon, Longview, WA). In January 2013, four females were sampled to determine the initial reproductive stage (pre-treatment, time 0). Fish were anesthetized by immersion in tricaine methanesulfonate (MS-222, Argent, Redmond, WA) and body weight (BW) and fork length (FL) recorded (1187.4 ± 49.4 g BW, 46.9 ± 0.5 cm FL). Blood (1 ml) was collected from the caudal vasculature with heparinized syringes and placed in glass tubes on crushed ice. Plasma was obtained by centrifugation of whole blood (3000g, 15 min, 4 °C) and stored at 20 °C for sex steroid (T, E2 and 11KT) analyses by enzyme-linked immunosorbent assays (ELISAs). Anesthetized fish were euthanized by decapitation for the collection of tissues. Pituitary glands were collected, immediately frozen in liquid nitrogen and stored at 80 °C until total RNA extraction for gonadotropin subunits (fshb, lhb and gpa) and drd2 gene expression analyses by qPCR. Ovaries and liver were removed and weighed for calculation of the gonadosomatic index (GSI = gonad weight 100/BW) and hepatosomatic index (HSI = liver weight 100/BW). For histology, a middle portion of the ovary was preserved in Bouin’s fixative for 24 h before storage in 70% ethanol. Subsequently, 17 females from the same cohort (1252.4 ± 44.3 g BW, 47.7 ± 0.5 cm FL) were randomly distributed into four groups (n = 4–5 fish/group) and implanted intramuscularly with sustained-release cholesterol pellets (30 mg, 95% cholesterol and 5% cellulose) containing T at doses of 0 (control), 0.15, 0.75 or 3.75 mg. Blood (0.7 ml) was collected on days 3, 7, 14, 21 and 28 after being implanted and the time course of sex steroids was determined. On day 28, all fish were euthanized as described above, and tissues (blood, pituitary, ovary and liver) were collected to examine the effect of different doses of T on the pituitary–ovary axis. Males in the tank were also treated with T and sampled as described for females, but results are not included in this study. Before study 2 was initiated, the release kinetics from the sustained-release cholesterol pellets containing T was examined in vitro. Briefly, implants containing 0, 0.15, 0.75 and 3.75 mg of
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T (n = 4 implants/dose) were placed in a 48-well plate (one implant/well) containing 500 ll of in vitro buffer (ELISA buffer, 0.1 M potassium buffer, pH 7.4). The plate was placed on a rocking shaker in a controlled-temperature incubator at 12 °C. On day 1, 3, 7, 14, 21 and 28, the incubation buffer was taken from each well and stored at 30 °C until analysis of T by ELISA. The buffer was refreshed after each sampling. In vitro release profiles are shown in Supplementary Fig. 1. 2.1.3. Study 3: effect of long-term T, GnRHa and the dopamine D2receptor antagonist, Met, treatments on the pituitary–ovary axis of prepubertal female sablefish F1 sablefish (broodyear 2010) were produced at the Manchester Research Station and held in net-pens in Clam Bay until winter 2013. Water temperature in the net-pens fluctuated seasonally, reaching minimum values in February and maximum in August (average temperature was 10.9 ± 0.2 °C; 7.7 °C min. and 14.9 °C max. corresponding to February and August 2012, respectively). In January 2013, fish were transported to three different outdoor tanks (4 m3) supplied with flow-through, sand-filtered and UVtreated seawater. They had been previously implanted with passive-integrated transponder tags and a blood sample taken so that sex could be genetically determined using genomic DNA from red blood cells (Rondeau et al., 2013). Only females were selected for this study, and exposed to natural photoperiod and water temperature. During the experiment, the water temperature increased naturally from 10.4 to 13.5 °C (average temperature for this period was 12.7 ± 0.2 °C) from 15 May to 18 July. Fish were fed ad libitum on dry pellets. In May 2013 (coinciding with the onset of the ovarian vitellogenic growth in the wild, Guzmán et al., unpublished), six 3-year old F1 female sablefish were sampled to determine the initial stage (pre-treatment, time 0). BW and FL were recorded (3411.3 ± 193.8 g BW, 62.7 ± 1.3 cm FL) and tissues (blood, pituitary, ovary and liver) were collected. On the same day, 48 females from the same cohort were anesthetized, BW and FL recorded (3649.6 ± 54.2 g BW, 64.8 ± 0.3 cm FL) and implanted every 3 weeks over 9 weeks with intramuscular, sustained-release cholesterol pellets containing: blank (control), T (10 mg), GnRHa ([des-Gly10, D-Ala6]-LH-RH, 200 lg) and Met (10 mg) alone or in combination. Every 3 weeks (coinciding with the next implantation), blood (1 ml) was collected from all fish to track levels of sex steroids. Three weeks after receiving the last implant, all fish were euthanized and tissues collected to examine the effect of T, GnRHa and Met, alone or in combination. 2.1.4. Study 4: steroidogenic capability of ovaries of prepubertal sablefish in vitro The same group of F1 female sablefish (broodyear 2003) that was used for study 1, and previously employed to characterize the reproductive endocrine axis in this species (Guzmán et al., 2013), was used for study 4. In August 2012, five 9 year-old females (3190 ± 124 g BW, 64.3 ± 8.9 cm FL) were sampled to obtain ovaries for in vitro culture. The ovaries were cut into 40 mg pieces and distributed into 24-well polystyrene culture plates so that each well received one piece of the ovaries from an individual fish. Culture wells contained 1 ml of Cortland’s solution supplemented with 0.2% BSA. An ovarian subsample was also collected for histology. Tissues were pre-incubated at 12 °C with gentle orbital shaking at 100 rpm for 1 h. After the pre-incubation, the medium was removed and replaced with either fresh medium alone (control) or medium containing T (1, 10 and 100 ng/ml), Met (10, 100 and 1000 lM), GnRHa (10, 100, 1000 nM), human chorionic gonadotropin (hCG, 0.1, 1 and 10 IU/ml) or pregnant-mare serum gonadotropin (PMSG, 0.1, 1 and 10 IU/ml). The highest doses of T
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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(100 ng/ml), Met (1 mM) and hCG (10 IU/ml) were also tested in combination. GnRHa and PMSG were not included in the combination because in previous ovarian incubations we observed no steroidogenic effect of GnRHa, while the effect of PMSG was similar to hCG. After a 24-h incubation, the culture medium was collected and stored at 80 °C until sex steroid analysis by LC–MS/MS. 2.2. RNA isolation and cDNA synthesis Total RNA from sablefish tissues was isolated with Tri-Reagent (Molecular Research Center, Cincinnati, OH) using a TissueLyser II (Qiagen, Valencia, CA). For partial cDNA cloning and gene expression analysis, a total RNA subsample was diluted to 250 ng RNA/ll in nuclease-free water and DNase treated using the Turbo DNA Free kit’s protocol (Ambion, Life Technologies, Grand Island, NY). RNA yields and quality were assessed by NanoDrop (ND1000 Spectrophotometer; NanoDrop Technologies, Rockland, DE) and gel electrophoresis. For RT, 500 ng of total RNA of each sample was reverse transcribed in 10-ll reactions with the Superscript II kit (Invitrogen, Life Technologies). Other necessary components for RT, such as random primers and RNase inhibitor, were purchased from Promega (Madison, WI). Negative control reactions were performed without the addition of the RT enzyme for a subset of the RNA samples. 2.3. Quantitative real-time PCR Quantitative PCR assays were run on an ABI 7900HT Fast RealTime PCR System (Life Technologies) in 384-well plates using standard cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Reactions were 12.5 ll each and consisted of 1 Power SYBR Green master mix (Applied Biosystems, Life Technologies), 150 nM of the forward and reverse primer (Supplementary Table 1), and 0.5 ng cDNA template based on the amount of total RNA loaded into the RT reactions. Standard curves were generated from serial dilution of cDNA pooled from RNA samples obtained from the target tissue (i.e., brain, pituitary, or ovary; one RNA sample representative of each group or treatment was included in the standard). Standard curve samples ranged from 0.1 to 5 ng cDNA, based on the amount of RNA added to the RT reactions, and represented 4 dilutions over this range. Linearity of the standard curve was confirmed for the assays and results were analyzed using the relative standard curve method. Each standard curve cDNA dilution was run in triplicate, while each sample was run in duplicate. For each gene, within a study, all samples were assayed in the same plate to avoid across plate variation. A dissociation or melt curve analysis step was included in each qPCR to confirm the assay specificity. Negative controls included in each plate consisted of either no cDNA template or RNA that was not reverse transcribed. These controls showed no detectable amplification over 40 cycles of PCR. Results for each target gene were normalized to eef1a which was demonstrated to be stable across groups in all tissues (results not shown). To improve the presentation of results, the mean value of the wild maturing (study 1) or time-0 fish (study 2 and 3) was set to 1. 2.4. Sex steroid analyses 2.4.1. Steroid analysis by ELISA In the in vivo studies (study 2 and 3), as well to determine the in vitro release of T loaded in the cholesterol pellets (study 2), levels of sex steroids were quantified by ELISA using protocols validated for sablefish (Guzmán et al., 2013). The sensitivities of the assays, calculated as maximum binding minus twice the standard
deviation, were 11.9, 2.8 and 4.9 pg/ml for the E2, T and 11KT immunoassays, respectively. The ED50s for the E2, T, and 11KT assays were 1.32, 0.15 and 0.07 ng/ml, respectively. Intra-assay coefficients of variation (CVs; n = 10) were 5.7%, 4.1% and 3.7% for E2, T and 11KT, respectively. Inter-assay CVs were 7.3%, 13.7% and 11.2% for E2, T and 11KT, respectively. Other details on cross-reactivity of E2, T and 11KT antibodies, and correlation between levels of E2, T and 11KT measured by ELISA and LC–MS/ MS in plasma samples of sablefish are provided in Supplementary Figs. 2–4. 2.4.2. Steroid analysis by LC–MS/MS Levels of sex steroids in ovarian incubation media (study 4) were determined by LC–MS/MS using a modified method described previously (Koren et al., 2012). Samples were extracted and cleaned using solid-phase extraction. A 250 ll aliquot of culture medium was diluted with water (1:1, v/v), followed by the addition of 20 ll of a mixture of surrogate standards (S-std) containing 11b-hydroxyandrostendione-d7, 17b-estradiol-d4 and progesterone-d9 in methanol (250 ng/ml), 100 ll of glacial acetic acid and 1 ml of water. The solution was mixed and loaded into a 3 ml Strata-X reversed-phase cartridge containing 60 mg of sorbent (Phenomenex Inc, Torrance, CA, USA) previously conditioned with 2 ml of methanol, followed by the addition of 2 ml of water. After the sample was loaded, the cartridge was washed with a mixture of methanol/water (60/40, v/v). The cartridge was dried under vacuum for 30 min and the target steroids were eluted with 1.5 ml of methanol into clean 2-ml vials. The volume of the final extract was reduced to 0.5 ml under a stream of nitrogen gas at room temperature. Estrone-d4 was added as an internal standard (IS; 20 ll of a 250 ng/ml methanolic solution) and an aliquot was transferred into a vial for injection into the LC– MS/MS. The sample extracts were analyzed by LC (Acquity system, Waters Co., Milford, MA) coupled with a triple quadrupole tandem mass spectrometer (MS/MS, QTRAP 5500, AB Sciex, Framingham, MA). For each sample, 10 ll of extract was injected onto the LC that was equipped with a 0.2-lm pre-filter followed by a 2.1 5.0 mm (1.7-lm particle size) C18 guard column and a 2.1 150 mm (1.7 lm particle size) reversed-phase column. Water (solvent A) and methanol (solvent B) were used as the mobile-phase. The total analysis time was 30 min using a linear gradient, as follows (solvent A/solvent B): initial gradient was 60/40 at 0.2 ml/min; 14 min to 36/64 at 0.2 ml/min; 5 min to 20/80 at 0.2 ml/min; 0.1 min to 100% solvent B at 0.2 ml/min; 0.1 min to increase the flow up to 0.35 ml/min and held for 4.8 min; 0.1 min to reduce flow to 0.30 ml/min; 0.9 min to initial gradient 60/40 at 0.3 ml/ min and held for 5 min. The column temperature was maintained at 45 °C. Electrospray ionization (ESI) mode was used for the ionization of all analytes. The MS/MS was operating in both negative and positive ion mode and the steroids were detected via multiple-reaction monitoring (MRM). The ion source was kept at 700 °C and capillary voltage varied between 4.5 kV (negative mode) and 5.5 kV (positive mode). Other details on the MRM parameters are given in the Supplementary Table 2. The analytes were quantified by S-std and based on the calibration curve of each analyte. The recovery of each S-std was calculated using the internal standard. The LC–MS/MS method used to measure steroids was validated by spiking the analytes at 0.1 ng/ml into culture medium before and after the extraction/cleanup on the solid-phase extraction. Using this approach, the recovery of steroids and any potential matrix effect were evaluated. The percent recovery for all analytes in both tests varied from 70% to 120%. Spiking solutions contained all of the target analytes as well as deuterated surrogate and internal standards used for quantifying the steroids
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and calculating the recovery of the surrogate standards. A method blank (LC–MS grade water), a spiked blank and a spiked matrix sample were analyzed with each sample batch. The percent recoveries of the steroids in the spiked samples ranged from 81% to 116%. The recoveries of the surrogate standards in both culture medium samples and associated quality assurance samples ranged from 67% to 107%. The LC–MS/MS system was calibrated using 5 different levels of steroids ranging from 0.03 to 30 ng/ml (in methanol) and surrogate standards were used to calculate the analyte concentrations. Another methanolic standard solution (LC-QC) was injected multiple times before, during and after each sequence of sample extracts in order to measure the stability of the instrument response. The relative standard deviation for the LC-QC injections within each sequence was less than 15% for all analytes. Limit of quantitation was compound dependent and varied from 0.02 to 0.6 ng/ml.
2.5. Histology and image analysis Fixed ovaries were dehydrated through a graded series of ethanol and embedded in paraffin wax, sectioned at a thickness of 4 lm, and stained with hematoxylin and eosin. Microscope images were captured using a Nikon (Melville, NY) video camera and analyzed through NIS Elements image software version 2.3 (Nikon). For analysis, the 30 largest follicles contained within a frame of 3.5 mm2 were selected and their areas measured by NIS Elements image software. Thirty follicles were measured in three non-overlapping frames per section. The radius (r) was calculated from the circumference and used to calculate follicle volume (V) according to the formula: V = 4/3pr3. We defined the maximum ovarian follicle volume per female as the average volume of the largest follicles (n = 90).
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2.6. Statistical analyses Statistical analyses were performed using Prism 5 software for Mac OSX (GraphPad Software, La Jolla, CA) with the minimum level of significance set to P < 0.05. In study 1, differences between prepubertal F1 and maturing females were assessed with a Student’s t-test. In study 2 and 3, differences in the in vitro release of T loaded in the cholesterol pellets, and in plasma sex steroid levels were analyzed using a two-way ANOVA with dose/treatment and time as independent variables and where significant differences were observed, Bonferroni post-tests were conducted. All other parameters analyzed in study 2–4 were assessed by oneway ANOVA and where significant differences were observed, Tukey multiple comparisons tests were conducted. For statistical purposes, in study 4, the lowest detectable value was assigned to samples below the limit of quantitation. When necessary to normalize distribution, values were Log-transformed prior to the ANOVA. Data are expressed as mean ± standard error of the mean (SEM).
3. Results 3.1. Study 1: tissue distribution of sablefish drd2 transcripts and comparison of expression levels in brain, pituitary and ovary between prepubertal and maturing females Transcripts for drd2 were found in all the reproductive tissues examined in sablefish, including brain, pituitary, ovary and testis, as well as in heart and spleen (Fig. 1). Levels of drd2 transcripts were highest in the brain, ovary and testis. Both brain and pituitary transcript levels of drd2 were higher in prepubertal F1 females relative to wild maturing females (4- and
Fig. 1. Tissue distribution of dopamine receptor D2 mRNA (drd2) in sablefish (upper) and its relative expression in brain, pituitary and ovary obtained from wild maturing (filled columns) and prepubertal F1 female sablefish (open columns) (lower). Transcript levels of eef1a were assessed to verify quality and equal loading of cDNAs. Gene expression levels were determined by qPCR and normalized to eef1a. Data are expressed as the mean ± SEM (n = 3). Columns with an asterisk are significantly different (t-test, p < 0.05).
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 2. Effect of different doses of testosterone on the pituitary–ovary axis in F1 female sablefish. Females (1.25 ± 0.04 kg) were treated with a single implant containing 0.15, 0.75 or 3.75 mg of T for 28 days. (A) Plasma profile of sex steroid levels, determined by ELISA. (B) Pituitary gene expression levels of gonadotropin subunits and (C) dopamineD2 receptors, determined by qPCR and normalized to eef1a. (D) Representative micrographs of ovaries from control (1) or females treated with 0.75 mg of T (2) after 28 days of treatment. (E) Maximum ovarian follicle volume, calculated by measuring the maximum area of the most advanced follicles (n = 90) per female using NIS-Elements Version 2.3. Data are expressed as the mean ± SEM (n = 4–5). Values with different superscript letters are significantly different, whereas asterisks (⁄) indicate significant differences from time 0 (ANOVA, p < 0.05).
30-fold respectively), whereas no significant differences were observed for drd2 transcripts in the ovary (Fig. 1). 3.2. Study 2: effect of different doses of T on the pituitary–ovary axis of prepubertal females The effects of sustained-release cholesterol pellets containing T at doses of 0.15, 0.75 and 3.75 mg on plasma levels of sex steroids (T, E2 and 11KT) in prepubertal F1 female sablefish for 28 days are shown in Fig. 2A. The low dose of T (0.15 mg) did not affect plasma levels of T or E2 but increased plasma 11KT levels at day 7 and 14. The mid dose of T (0.75 mg) increased levels of plasma T for 14 days and 11KT at days 7, 14 and 21, but did not increase E2 levels relative to control. In contrast, the high dose of T (3.75 mg) induced significant increases in plasma T levels for 28 days, and both E2 and 11KT for 21 days. At the pituitary level, sustained administration of T did not significantly affect transcript levels for fshb or gpa relative to control, although the low dose of T (0.75 mg) increased transcripts for fshb relative to time 0 (Fig. 2B). The highest dose of T (3.75 mg) induced a significant increase in lhb transcripts (elevated 35-fold relative to
control fish). Treatments with T did not affect pituitary drd2 transcripts (Fig. 2C). As expected, the ovaries of females at the start of the study (time 0, data not shown) were characterized by the presence of follicles in primary growth (perinucleolus stage). Treatments with T did not stimulate the initiation of secondary oocyte growth (i.e., the appearance of cortical alveoli or yolk granules), and ovarian follicles remained at the perinucleolus stage (Fig. 2D). However, treatments with mid and high doses of T induced a significant increase in the maximum volume of ovarian follicles after 28 days of treatment (Fig. 2E). No effect of treatments on GSI or HSI was observed (Supplementary Fig. 5). 3.3. Study 3: effect of long-term T, GnRHa and the dopamine D2receptor antagonist, Met, treatments on the pituitary–ovary axis of prepubertal female sablefish The effects of sustained-release cholesterol pellets containing T (10 mg), GnRHa (200 lg) and Met (10 mg) alone or in combination on plasma levels of sex steroids (T, E2 and 11KT) in prepubertal F1 female sablefish for 9 weeks are shown in Fig. 3A. Only T-
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 3. Effect of testosterone (T), metoclopramide (Met) and gonadotropin-releasing hormone agonist (GnRHa) on the pituitary–ovary axis in F1 female sablefish. Fish (3.65 ± 0.05 kg) were treated every 3 weeks with implants containing T (10 mg), Met (10 mg) or GnRHa (200 lg) alone or combined, for 9 weeks. (A) Plasma profile of sex steroid levels, determined by ELISA. (B) Pituitary gene expression levels of gonadotropin subunits and (C) dopamine-D2 receptors, determined by qPCR and normalized to eef1a. (D) Representative micrographs of ovaries from control females (1) or those treated with T (2), Met (3) or GnRHa (4) after 9 weeks. (E) Ovarian follicle volume, calculated by measuring the maximum area of the most advanced follicles (n = 90) per female using NIS-Elements software. Data are expressed as the mean ± SEM (n = 6). Values with different superscript letters are significantly different, whereas asterisks (⁄) indicate significant differences from time 0 (ANOVA, p < 0.05).
containing implants increased plasma levels of sex steroids. Specifically, T and 11KT were maintained at elevated levels over the 9 week experiment, while levels of E2 were elevated in females treated with T alone or combined with Met and GnRHa at week 3, and in those treated with T combined with Met and/or GnRHa at week 9. At the pituitary level, sustained administration of Met (alone) increased the expression of fshb, but had no effect on lhb or gpa transcripts (Fig. 3B). When Met was combined with T and/or GnRHa the fshb transcripts were not significantly different than controls. Both T and GnRHa significantly affected the transcript levels of lhb (70- and 7-fold respectively), and the combination of T and GnRHa had a strong synergistic effect on the expression of lhb (2000-fold higher than control). This combination of T and GnRHa was also effective in elevating transcripts of gpa, and the effect was enhanced when Met was included. Conversely, pituitary drd2 transcripts were not affected by any of the treatments (Fig. 3C). The ovaries of females were also characterized by the presence of follicles in primary growth (perinucleolus stage) at time 0 (results not shown). None of the treatments tested induced the
transition to secondary oocyte growth (Fig. 3D). However, as also observed in study 2, administration of T was effective in elevating the maximum ovarian follicle volume relative to controls (Fig. 3E). A slight but significant effect on the maximum ovarian follicle volume was also observed in females treated with Met (Fig. 3E), although the maximum ovarian follicle volume in controls was lower than that of fish sampled at time 0 and Met did not significantly increase follicle volume over fish at time 0. No significant effects of treatments on GSI or HSI were observed (Supplementary Fig. 6). 3.4. Study 4: steroidogenic capability of primary follicles in vitro Effects of T, Met and hCG alone and in combination on the in vitro ovarian production of various sex steroids is shown in Fig. 4. From a total of 15 steroids simultaneously analyzed by LC–MS/MS (see complete list in Supplementary Table 2), only 8 of them were detectable in the culture medium. These included 17a-hydroxyprogesterone (17OHP4), androstenedione (A4), 11bhydroxyandrostenedione (11OHA4), 11b-hydroxytestosterone (11OHT), T, E2, 5a-dihydrotestosterone (DHT) and 11KT.
Please cite this article in press as: Guzmán, J.M., et al. Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.02.024
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Fig. 4. In vitro production of sex steroids by perinucleolus stage ovarian fragments of F1 sablefish exposed to testosterone (100 ng/ml), metoclopramide (1 mM) and human chorionic gonadotropin (hCG, 10 IU/ml) alone or combined. Ovarian fragments (40 mg) were cultured in Cortland’s solution for 24 h at 12 °C. Data are expressed as the mean ± SEM (n = 5). Values with different superscript letters are significantly different (ANOVA, p < 0.05). For statistical purposes, the lowest detectable value was assigned to samples below the detection limit (indicated by a dotted line). In these cases, asterisks (⁄) indicate significant differences from the control group (ANOVA, p < 0.05).
As expected, the ovaries used for this study were also at the perinucleolus stage. Only E2 was detected in the culture medium of control samples (ranged from 0.06 to 0.13 ng/ml). In general, T was the most potent treatment in increasing the production of A4, 11OHA4, 11OHT, E2, DHT and 11KT (Fig. 4 and Supplementary Fig. 7). Ovarian fragments incubated with the highest dose of T (100 ng/ml) preferentially produced A4 (>10 ng/ml) and DHT (>5 ng/ml) over other metabolites (11OHA4, 11OHT, E2, 11KT,
