RESEARCH ARTICLE Molecular Reproduction & Development 82:651–662 (2015)
Identification of microRNAs Associated With Sexual Maturity in Rainbow Trout Brain and Testis Through Small RNA Deep Sequencing RODOLFO FARLORA, DIEGO VALENZUELA-MIRANDA, PAMELA ALARCON-MATUS, AND CRISTIAN GALLARDO-ESCARATE* Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), n, Concepcio n, Chile Department of Oceanography, University of Concepcio
SUMMARY MicroRNAs are ubiquitous, short, non-coding RNA molecules that function as posttranscriptional regulators of gene expression in a variety of biological processes. In order to further our understanding of the microRNA hierarchy in the reproductive axis of male teleosts, four small RNA libraries were constructed, and sequenced from the testis and brain of sexually mature adults and immature juvenile rainbow trout (Oncorhynchus mykiss) using Illumina small-RNA deep sequencing. We obtained 56,632,987; 39,870,661; 82,454,370; and 53,143,465 high-quality, filtered reads for immature testis, mature testis, immature brain, and mature brain, respectively. Among the libraries, 433 known mature piscine miRNAs were identified, with 124 and 116 significantly differentially expressed miRNAs found between sexually immature/mature testes and immature/mature brain tissues, respectively. Among these differentially expressed miRNAs, miR-129, -130c, -135b, -140, -146a, -192, -199a, -16, and, -19b showed higher abundance in the testis whereas miR-10b, -183, -199, -375, -1937, and miR-nov210 were more abundant in the brain. In silico target prediction of these differentially expressed miRNAs suggested their putative roles in the sexual maturation of male rainbow trout. Mol. Reprod. Dev. 82: 651662, 2015. ß 2015 Wiley Periodicals, Inc. Received 19 November 2014; Accepted 29 April 2015
INTRODUCTION Sexual maturation involves profound hormonal, cellular, and behavioral changes that lead to an individual’s ability to produce viable gametes. In fish, the age at which maturation occurs is determined by several internal and external factors, including photoperiod, water temperature, food availability, rainfall, somatic growth, and gonadal status (Okuzawa 2002). Under aquaculture conditions, phenotypic responses to improved growth conditions and feed availability often with associated higher adiposity and energy storesare the probable major initiators of early puberty commonly observed in many fish (Taranger et al., 2010).
ß 2015 WILEY PERIODICALS, INC.
Corresponding author: Universidad de Concepcion P.O. Box 160-C Concepcion, Chile E-mail: [email protected]
Grant sponsor: FONDECYT from CONICYT-Chile; Grant number: 3130446; Grant sponsor: FONDAP from CONICYT-Chile; Grant number: 15110027
Published online 15 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22499
Precocious maturity in farmed fish can pose negative consequences in several productive traits, however, including growth rate, food-conversion rate, survival rates, agonistic behavior, and general welfare (Taranger et al., 2010). As in other animals, puberty is thought to activate the brain-pituitary-gonad axis, but the details of this process in teleosts are unclear.
Abbreviations: miRNAs, microRNAs; RT-qPCR, reverse-transcriptase quantitative PCR; UTR, untranslated region
Molecular Reproduction & Development
FARLORA
MicroRNAs (miRNAs) are ubiquitous, short, noncoding RNA molecules that function as post-transcriptional regulators of gene expression across taxa. miRNAs participate in a variety of biological processes, including development, organogenesis, signal transduction, and disease (Ambros 2004; Bushati and Cohen 2007; Begemann 2008; Lu et al., 2008; Mudhasani et al., 2008). The use of deep-sequencing technologies has dramatically accelerated the discovery of new miRNAs due to its throughput and accuracy compared with traditional methods, allowing the detection of novel, and low-abundance miRNAs. In males, the onset of puberty is characterized by the initiation of spermatogenesis. Several recent studies in mammals have revealed a regulatory role of miRNAs in spermatogenesis (Tong et al., 2011; Smorag et al., 2012; Tong et al., 2012; Van den Driesche et al., 2014; Wu et al., 2014); in contrast, information supporting a role for miRNAs during sexual maturation in teleosts is scarce. Knowledge about miRNAs and their target genes may help develop novel methods to control puberty and to improve the performance of economically important traits in fish. Before their biological functions can be assessed, however, the miRNAs must first be identified and annotated. Previous studies in Atlantic halibut (Hippoglossus hippoglossus) found several miRNAs up-regulated in adult testis as compared to adult ovariesincluding let-7a, miR-143, miR-145, and miR-202-3pwhereas miR-451 is significantly down-regulated in the brain of juveniles as compared to adult females (Bizuayehu et al., 2012). In Nile tilapia (Oreochromis niloticus), miR-129-3p and miR-727-3p were significantly up-regulated in mature females as compared to mature males, whereas miR-132a and miR-212 were more abundant in mature males as compared to females (Xiao et al., 2014). miRNA profiling in rainbow trout (Oncorhynchus mykiss) revealed 13 miRNAs with differential patterns of expression during ovarian development (Juanchich et al., 2013), while other miRNA populations were reported from somatic tissues (Salem et al., 2010) and eggs (Ma et al., 2012). The objective of the present study was to build on these previous studies by characterizing miRNA expression and identifying the potential targets of these miRNAs in brain and testicular tissues from sexually immature and mature rainbow trout males.
ET AL.
RESULTS Assessment of Gonad Development In order to evaluate the testicular development of the individuals used in the transcriptomic study, a small piece of each testis was dissected and processed for histological analysis. The selected immature juvenile males testes (stages IIIII) (Gomez et al., 1999) exhibited an incomplete germ line that was characterized by the presence of small gonad acini containing mitotic germ cells, which consisted of differentiating spermatogonia with few primary spermatocytes (Fig. S1A, C, and E). Selected mature adult testes (stage VIII) (Gomez et al., 1999), on the other hand, exhibited fused gonad acini, containing mainly spermatozoa (Fig. S1B, D, and F). Mean body weight (g) and gonadosomatic index values (%) for immature juveniles (n ¼ 5) were 231.82 56.25 g and 0.05 0.01% (mean standard deviation), respectively; for mature adults (n ¼ 5), values were 3011.80 542.02 g and 1.55 0.44%, respectively (Table S1).
Sequence Analysis of miRNAs in Rainbow Trout Testis and Brain Illumina small RNA deep sequencing generated 56,632,987; 39,870,661; 82,454,370; and 53,143,465 high-quality filtered reads for immature testis, mature testis, immature brain, and mature brain, respectively (Table 1). The size distribution of these mapped reads followed a similar trend for the four libraries, with the majority of reads measuring between 19 and 24 nt (Fig. 1). The most abundant size class was 22 nt, accounting for 58.54%, 50.91%, 57.40%, and 57.83% of the total reads in immature-testis, mature-testis, immature-brain, and mature-brain libraries, respectively. For all libraries, the majority of unique sequences for mature miRNA exhibited low counts (
Identification of microRNAs Associated With Sexual Maturity in Rainbow Trout Brain and Testis Through Small RNA Deep Sequencing RODOLFO FARLORA, DIEGO VALENZUELA-MIRANDA, PAMELA ALARCON-MATUS, AND CRISTIAN GALLARDO-ESCARATE* Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), n, Concepcio n, Chile Department of Oceanography, University of Concepcio
SUMMARY MicroRNAs are ubiquitous, short, non-coding RNA molecules that function as posttranscriptional regulators of gene expression in a variety of biological processes. In order to further our understanding of the microRNA hierarchy in the reproductive axis of male teleosts, four small RNA libraries were constructed, and sequenced from the testis and brain of sexually mature adults and immature juvenile rainbow trout (Oncorhynchus mykiss) using Illumina small-RNA deep sequencing. We obtained 56,632,987; 39,870,661; 82,454,370; and 53,143,465 high-quality, filtered reads for immature testis, mature testis, immature brain, and mature brain, respectively. Among the libraries, 433 known mature piscine miRNAs were identified, with 124 and 116 significantly differentially expressed miRNAs found between sexually immature/mature testes and immature/mature brain tissues, respectively. Among these differentially expressed miRNAs, miR-129, -130c, -135b, -140, -146a, -192, -199a, -16, and, -19b showed higher abundance in the testis whereas miR-10b, -183, -199, -375, -1937, and miR-nov210 were more abundant in the brain. In silico target prediction of these differentially expressed miRNAs suggested their putative roles in the sexual maturation of male rainbow trout. Mol. Reprod. Dev. 82: 651662, 2015. ß 2015 Wiley Periodicals, Inc. Received 19 November 2014; Accepted 29 April 2015
INTRODUCTION Sexual maturation involves profound hormonal, cellular, and behavioral changes that lead to an individual’s ability to produce viable gametes. In fish, the age at which maturation occurs is determined by several internal and external factors, including photoperiod, water temperature, food availability, rainfall, somatic growth, and gonadal status (Okuzawa 2002). Under aquaculture conditions, phenotypic responses to improved growth conditions and feed availability often with associated higher adiposity and energy storesare the probable major initiators of early puberty commonly observed in many fish (Taranger et al., 2010).
ß 2015 WILEY PERIODICALS, INC.
Corresponding author: Universidad de Concepcion P.O. Box 160-C Concepcion, Chile E-mail: [email protected]
Grant sponsor: FONDECYT from CONICYT-Chile; Grant number: 3130446; Grant sponsor: FONDAP from CONICYT-Chile; Grant number: 15110027
Published online 15 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22499
Precocious maturity in farmed fish can pose negative consequences in several productive traits, however, including growth rate, food-conversion rate, survival rates, agonistic behavior, and general welfare (Taranger et al., 2010). As in other animals, puberty is thought to activate the brain-pituitary-gonad axis, but the details of this process in teleosts are unclear.
Abbreviations: miRNAs, microRNAs; RT-qPCR, reverse-transcriptase quantitative PCR; UTR, untranslated region
Molecular Reproduction & Development
FARLORA
MicroRNAs (miRNAs) are ubiquitous, short, noncoding RNA molecules that function as post-transcriptional regulators of gene expression across taxa. miRNAs participate in a variety of biological processes, including development, organogenesis, signal transduction, and disease (Ambros 2004; Bushati and Cohen 2007; Begemann 2008; Lu et al., 2008; Mudhasani et al., 2008). The use of deep-sequencing technologies has dramatically accelerated the discovery of new miRNAs due to its throughput and accuracy compared with traditional methods, allowing the detection of novel, and low-abundance miRNAs. In males, the onset of puberty is characterized by the initiation of spermatogenesis. Several recent studies in mammals have revealed a regulatory role of miRNAs in spermatogenesis (Tong et al., 2011; Smorag et al., 2012; Tong et al., 2012; Van den Driesche et al., 2014; Wu et al., 2014); in contrast, information supporting a role for miRNAs during sexual maturation in teleosts is scarce. Knowledge about miRNAs and their target genes may help develop novel methods to control puberty and to improve the performance of economically important traits in fish. Before their biological functions can be assessed, however, the miRNAs must first be identified and annotated. Previous studies in Atlantic halibut (Hippoglossus hippoglossus) found several miRNAs up-regulated in adult testis as compared to adult ovariesincluding let-7a, miR-143, miR-145, and miR-202-3pwhereas miR-451 is significantly down-regulated in the brain of juveniles as compared to adult females (Bizuayehu et al., 2012). In Nile tilapia (Oreochromis niloticus), miR-129-3p and miR-727-3p were significantly up-regulated in mature females as compared to mature males, whereas miR-132a and miR-212 were more abundant in mature males as compared to females (Xiao et al., 2014). miRNA profiling in rainbow trout (Oncorhynchus mykiss) revealed 13 miRNAs with differential patterns of expression during ovarian development (Juanchich et al., 2013), while other miRNA populations were reported from somatic tissues (Salem et al., 2010) and eggs (Ma et al., 2012). The objective of the present study was to build on these previous studies by characterizing miRNA expression and identifying the potential targets of these miRNAs in brain and testicular tissues from sexually immature and mature rainbow trout males.
ET AL.
RESULTS Assessment of Gonad Development In order to evaluate the testicular development of the individuals used in the transcriptomic study, a small piece of each testis was dissected and processed for histological analysis. The selected immature juvenile males testes (stages IIIII) (Gomez et al., 1999) exhibited an incomplete germ line that was characterized by the presence of small gonad acini containing mitotic germ cells, which consisted of differentiating spermatogonia with few primary spermatocytes (Fig. S1A, C, and E). Selected mature adult testes (stage VIII) (Gomez et al., 1999), on the other hand, exhibited fused gonad acini, containing mainly spermatozoa (Fig. S1B, D, and F). Mean body weight (g) and gonadosomatic index values (%) for immature juveniles (n ¼ 5) were 231.82 56.25 g and 0.05 0.01% (mean standard deviation), respectively; for mature adults (n ¼ 5), values were 3011.80 542.02 g and 1.55 0.44%, respectively (Table S1).
Sequence Analysis of miRNAs in Rainbow Trout Testis and Brain Illumina small RNA deep sequencing generated 56,632,987; 39,870,661; 82,454,370; and 53,143,465 high-quality filtered reads for immature testis, mature testis, immature brain, and mature brain, respectively (Table 1). The size distribution of these mapped reads followed a similar trend for the four libraries, with the majority of reads measuring between 19 and 24 nt (Fig. 1). The most abundant size class was 22 nt, accounting for 58.54%, 50.91%, 57.40%, and 57.83% of the total reads in immature-testis, mature-testis, immature-brain, and mature-brain libraries, respectively. For all libraries, the majority of unique sequences for mature miRNA exhibited low counts (
