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GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males
Animal Reproduction Science 139 (2013) 162–167
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males Marcio A. Figueiredo a , Raíssa V. Fernandes a , Ana L. Studzinski a , Carlos E. Rosa a , Carine D. Corcini b , Antônio S. Varela Junior a , Luis F. Marins a,∗ a b
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas – UFPEL, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 4 August 2012 Received in revised form 18 March 2013 Accepted 26 March 2013 Available online 4 April 2013 Keywords: Growth hormone Transgenesis Reproduction Zebrafish Trojan effect
a b s t r a c t Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growth hormone is a polypeptide produced and secreted by the adenohypophysis, promoting, as a main effect, somatic growth in vertebrates. Although the manipulation of GH gene by transgenesis have been showing promising results regarding fish growth (Devlin et al., 1994; Nam et al., 2001), its excess may lead to unwanted collateral effects. GH is well known by its role in many processes besides growth, having pleiotropic effects over morphology, physiology, metabolism, immunology, reproduction
∗ Corresponding author at: Universidade Federal do Rio Grande – FURG, Instituto de Ciências Biológicas, Av. Itália, Km 8, 96203-900 Rio Grande, RS, Brazil. Tel.: +55 53 32935191. E-mail address: [email protected] (L.F. Marins). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.03.012
and behavior (Devlin et al., 2006). Therefore, it is obvious that the maintenance of this hormone under supraphysiological levels may lead to a series of undesirable effects over other systems within the organism. Mori et al. (2007) have observed an alteration in the expression of genes related to immune and reproductive processes in amago salmon (Oncorhynchus masou). There were also observed a significant increase of metabolic rates and oxygen consumption in Atlantic salmon (Cook et al., 2000; Herbert et al., 2001) and tilapias (McKenzie et al., 2000, 2003), as a result of GH superexpression or its administration. In addition, GH-transgenesis in coho salmon decreases sperm quality in comparison to wild males (Fitzpatrick et al., 2011), and affects reproductive behavior during competition with hatchery males (Bessey et al., 2004). In the same way, Moreau et al. (2011) showed that GH-transgenic Atlantic salmon had lower overall fertilization success and
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reduced breeding performance relative to nontransgenics. Aiming to study the collateral effects caused by GH excess, our research group has developed a transgenic zebrafish (Danio rerio) model that overexpresses GH from the marine silverside fish Odontestes argentinensis (Figueiredo et al., 2007a). This lineage, named F0104, has been previously studied in terms of growth and gene expression from the somatotropic axis (Figueiredo et al., 2007b), metabolic rate, reactive oxygen species (ROS) production and aging (Rosa et al., 2008, 2010, 2011), intracellular signaling (Studzinski et al., 2009), muscular structure (Kuradomi et al., 2011) and salinity tolerance (Almeida et al., 2013). However, this model remains without an analysis concerning the reproductive parameters. Regarding reproduction, GH transgenesis may have a sex-dependant effect, once a sex-specific pulsatile secretion pattern of this hormone is already well characterized in mammals. In this vertebrate group, females present more frequent and less pronounced secretion peaks along the day, while males exhibit peaks, although less frequent, with larger amplitude (Jansson et al., 1985). Despite little information is available in fish, there is evidence that GH may also be secreted in an episodic fashion in carp (Zhang et al., 1994) and in a sex-dependant way in the rainbow trout (Gomez et al., 1996). Regarding transgenic zebrafish model, the object from the present study, the constant GH overexpression along time may induce a profile similar to that observed in females, thus leading to more pronounced effect in males. Considering that GH may interfere in the sexual maturation process and that transgenesis can alter the natural sex-dependant expression feature of this hormone, the present study aimed to evaluate the reproductive parameters of transgenic adult zebrafish males from F0104 lineage. Two-years-old fish were used since it has been demonstrated that at this age, even at the end of reproductive cycle, sperm quality did not differ between young and old zebrafish (Kanuga et al., 2011). Therefore, the spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality and DNA integrity, comparing transgenic and non-transgenic fish. In addition, histological slides were made aiming to verify if transgenesis caused any effect over the tissue’s structure or the presence of characteristic cell types.
2. Materials and methods 2.1. Zebrafish Transgenic and non-transgenic zebrafish from lineage F0104 were kept in a closed system of water circulation containing 50 aquaria, with 15 l of volume each, oxygen levels near saturation, pH close to 7, photoperiod 14L:10D and temperature of 28 ◦ C. Animals were fed ad libitum, twice a day with a commercial fish food (Tetra ColorBits) enriched with alive nauplii of Artemia sp.
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2.2. Testes excision Fish at the same age (two years-old ± one month) were used in the procedures. The pair of testes, from 11 transgenic hemizygous males and 14 non-transgenic (control group), were excised and immediately placed in 100 L of Beltsville Thawing Solution (BTS) (Pursel and Johnson, 1975). Bathing in the aforementioned solution allows the collection of spermatozoa, which were not encysted, and keeps the tissue intact for further histological analyses. Following the bath, each gonad was fixed in buffered 4% formaldehyde solution during 24 h for the histological analyses. 2.3. Motility and the motility period of sperm Sperm motility and the motility period were evaluated in a microscope of phase-contrast using 1 L of zebrafish semen in BTS and 99 L of distilled water (25 ◦ C), intending to activate the spermatozoa. The motility period comprised, in seconds, the stage between the spermatozoa activation and the absence of progressive movement (a straight line movement). 2.4. Sperm membrane integrity Aiming to analyze the cytoplasmic membrane integrity of spermatozoa, two probes were used to allow differentiation between the two cellular types: intact and damaged. For evaluation, 5 L sperm sample was diluted in 20 L of working isotonic saline solution, including: 1.7 mM formaldehyde, 20 M carboxyfluorescein diacetate (CFDA), and 7.3 M propidium iodide (PI) (Varela Junior et al., 2012). Two hundred spermatozoa were analyzed and those presenting an intact membrane went through a hydrolysis process, transforming the CFDA into carboxyfluorescein, which accumulates in all compartments emitting green fluorescence as a singular characteristic. Spermatozoa presenting damaged membrane do not accumulate CFDA. In addition, damaged spermatozoa incorporate PI emitting a red fluorescence (Harrison and Vickers, 1990). 2.5. Mitochondrial functionality Intending to evaluate the mitochondrial sperm functionality, rhodamine probe 123 was used (Rh 123 – Sigma, Brazil). Sperm was evaluated after incubation of a 5 L sample with a 20 L Rh 123 solution (13 M) at 20 ◦ C for 10 min. Sperm with positive rhodamine staining (green fluorescence) were considered as having functional mitochondria. Conversely, nonfunctional mitochondria were characterized by negative rhodamine staining (sperm with no fluorescence). The rate of mitochondrial functionality was determined by the proportion of sperm emitting green fluorescence compared with total sperm (He and Woods, 2004). 2.6. DNA integrity Sperm DNA integrity was evaluated after putting a 10 L sperm sample in 10 L TNE (0.01 M Tris–HCl; 0.15 M NaCl;
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0.001 M EDTA; pH 7.2). After 30 s, 40 L of Triton solution 1× was added and, 30 s later, 10 L of acridine orange was added (2 mg/mL in deionized H2 O). The evaluation was done after 5 min, without exceeding 1 min of slide exposure. Sperm with green fluorescence were considered as having intact DNA, whereas those with red or orange fluorescence were considered as having denatured DNA (Bencharif et al., 2010). The frequency of DNA integrity was determined by the proportion of sperm emitting green fluorescence compared with the total number of sperm (green, red, or orange fluorescence) (Varela Junior et al., 2012).
3. Results
2.7. Histology
3.2. Mitochondrial functionality
Sexual maturation was examined by analysis of histological slides from testes. After 24 h immersed in buffered paraformaldehyde, the gonads were dehydrated by alcohol baths, with growing concentrations, followed by the processes of diaphanisation (xylol), impregnation and inclusion in Paraplast Xtra (P3808 – Sigma, Brazil), according to Carson and Hladik (2009). After inclusion, the material was sliced in a motorized rotary microtome (Leica RM2255) with thickness of 6 m. The fixed slides were stained with hematoxylin and eosin (Carson and Hladik, 2009). After the staining process, the material was visualized in an optic microscope under 100× magnification. Sexually mature testes presented the four cellular stages: spermatogonium, spermatid, spermatocyte and spermatozoid.
The spermatozoa from transgenic fish have presented a mean mitochondrial functionality of 51.1% (±5.4). The mean mitochondrial functionality of non-transgenic animals was 89.6% (±3.6), which was significantly higher than the observed in transgenic zebrafish (p < 0.05) (Fig. 1C).
3.1. Motility and motility period of spermatozoa Non-transgenic fish presented mean spermatic motility of 91.8% (±2.9), which was significantly higher (p < 0.05) than in transgenic individuals (36.4 ± 12.4%) (Fig. 1A). After verifying the percentage of motility, the motility period time was counted in seconds. The mean time observed until ceasing progressive movement of non-transgenic fish was 103.6 s (±12.9), which was significantly higher (p < 0.05) to mean motility period of transgenic fish (41.4 ± 12.7 s) (Fig. 1B).
3.3. Sperm membrane integrity Concerning the group of transgenic animals, the mean of fish presenting spermatozoa with intact membrane was 52.7% (±4.6). On the other hand, a significantly higher mean of 91.3% (±1.8) was found for non-transgenic fish presenting spermatozoa with intact membrane (p < 0.05) (Fig. 1D). 3.4. DNA integrity
2.8. Fertilization and hatching frequency Aiming to evaluate reproductive parameters, a breeding assessment was performed between non-transgenic couples (n = 5) and a crossbreeding between transgenic males and non-transgenic females (n = 5). Different females were used for each male. The eggs were collected in traps 1 h after spawning, intending to examine the fertilization rate in a stereoscopic microscope. Eggs characterized by 4–8 blastomeres were considered fertilized. Subsequently, all eggs were counted and incubated at 28 ◦ C until hatching. The hatching rate was determined 3 days after spawning and expressed as the percentage of hatched larvae in relation to total number of incubated eggs.
2.9. Statistical analyses The statistical analyses of GH transgenic and nontransgenic fish groups were realized using the software Statistix 9.0, Analytical Software (Tallahassee, FL, USA) (Statistix, 2008). Shapiro–Wilk test was run for analysis of the samples normality. The parameters considered with normal distribution were submitted to variance analysis, comparing transgenic and non-transgenic animals, followed by Tukey’s test HSD with significance level of 5%. The parameters without normal distribution were submitted to Kruskal–Wallis nonparametric test with significance level of 5%.
The group of transgenic fish presented significantly lower DNA integrity (p < 0.05) when compared with non-transgenic animals. The mean DNA integrity of spermatozoa from non-transgenic fish was 81.4% (±4.1), while the observed for transgenic fish was only 33.3% (±6.3) (Fig. 1E). 3.5. Fertilization and hatching frequency The results from fertilization and hatching are shown in Fig. 1F. The fertilization rate observed in non-transgenic fish was 90% (±1.4), while this parameter was significantly lower in transgenic individuals (76 ± 3.2%) (p < 0.05). The same tendency was observed concerning the hatching frequency. The percentage of hatched eggs from nontransgenic animals was 85% (±9.1) and from transgenic fish was 42% (±6.9) (p < 0.05). 4. Discussion The present study has shown that GH gene transgenesis produced a negative effect over the reproductive success in two-years-old zebrafish males from lineage F0104. All the analyzed parameters presented a significant decrease of sperm quality resulting in important consequences on the fertilization and spawning viability. These results may have important repercussions concerning the viability of the mentioned technology when used intending to increase
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Fig. 1. Comparison of spermatic parameters between GH-transgenic (T) and non-transgenic (NT) zebrafish males. (A) % of motile cells; (B) motility period (s); (C) % of functional mitochondria; (D) % membrane integrity; (E) % DNA integrity; (F) % fertilization and hatching. Asterisks represent statistically significant differences (p < 0.05).
production in aquaculture, as the amount of offspring generated by transgenic mates with such reproductive success could be a limiting factor. Besides, transgenic fish presenting decreased reproductive viability could represent a great environmental risk according to Trojan gene hypothesis, which postulates that a transgenic fish with advantage in sexual selection, associated with minor spawning viability, could extinguish an entire population of wild animals after a few generations (Muir and Howard, 1999). The first parameters analyzed in the present study were motility and motility period of sperm. Motility refers to spermatozoid capacity of moving toward the oocyte and it is a crucial factor for fertilization. Also, motility is activated by changes in intracellular ion concentrations, which are dependant of ATPases activity. These enzymes can alter the cellular ionic composition and produce a membrane hyperpolarization, inducing sperm motility (Schulz et al., 2010). ATPases are highly dependent on ATP and the
mitochondria play an important role within this mechanism (Bragadin et al., 2006). In the same sense, motility period is considered as the time in which the sperm keeps on movement and depends on the available energy. The obtained results concerning these two parameters indicate that spermatozoa from GH-transgenic fish present a significant diminishing of 60% approximately in relation to non-transgenic animals (Fig. 1A and B). These results are supported by the significant percentage decrease in functional mitochondria of 43% observed in transgenic individuals (Fig. 1C). Therefore, it is hypothesized that transgenic males possess a lower amount of available ATP, turning them less viable to fertilization. One possible explanation for decreased mitochondrial functionality could be the increased production of reactive oxygen species (ROS) induced by the excess of GH. Formerly, it was demonstrated that fish from lineage F0104 exhibit a significant increase in the metabolic rate and ROS generation in
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muscle, and decreased expression of genes from the antioxidant defense system (ADS) in liver and muscle (Rosa et al., 2008, 2011). The present scenario, with increased ROS associated with decreased ADS from testes, could represent an oxidative stress situation in which could cause great damages in the mitochondrial membrane and in its functionality (Sies and Jones, 2007). Thus, the spermatogonia exposed to elevated amount of ROS could be originating sperm with lower quantity of functional mitochondria and affecting of motility and motility period. The same way that elevated amount of ROS may alter the mitochondrial membrane, the spermatozoid membrane can undergo the same effect. The obtained result regarding the sperm membrane integrity has shown a significant diminishing of approximately 42% in transgenic fish (Fig. 1D). This decreasing may have an important effect over spermatozoa motility, once the membrane guides all water and ion exchanges between intra and extracellular environments and triggers the flagella movements (Alavi and Cosson, 2006). The plasmatic membrane of spermatozoa from teleost fish is a key component in gamete fusion, due the absence of acrosome in this vertebrate group, which directly affects fertilization (Bobe and Labbé, 2010). Besides membranes, ROS can also target the DNA molecule. Under a situation of oxidative stress, DNA may suffer strands damage, base removal or nucleotide modification, particularly in guanine rich regions. When these alterations are not repaired, they can cause serious damage to the cell and compromise its viability. In the present study, there was observed a significant reduction of approximately 60% in DNA integrity from transgenic animals (Fig. 1E). Although the DNA quality does not affects the spermatozoid’s fertilization capacity, this parameter is crucial for the embryo development (Zini et al., 2001; Boe-Hansen et al., 2008). Considering that all analyzed parameters indicate a significant diminishing of sperm quality from transgenic fish, a direct consequence over fertilization and hatching rates is expected. Fig. 1F summarizes the results of fertilization and hatching rates evincing significant decreases of 16 and 51%, respectively, even though the histological analysis have detected all characteristic cell types of mature testes in transgenic fish. These are concerning results when considering GH transgenesis application in aquaculture, if transgenic mates with such reproductive deficiency degree are not viable. These results are also concerning under an environmental point of view. Muir and Howard (1999) have developed a mathematic model that predicts extinction, after few generations, of wild populations in which transgenic fish presenting mating advantage associated with diminished viability of its offspring were introduced. However, this hypothesis is based on models built on data obtained under controlled laboratory conditions. Actually, within a variable environment, with low food availability and high competition at different levels, it is presumable that the transgenic animal will not achieve the same reproductive success from the wild-type and will be unable to spread its genes easily. Aiming to clarify this question, research involving reproductive behavior of transgenic individuals is made necessary to verify the existence of a possible
sexual selection advantage in relation to non-transgenic fish.
Acknowledgements This work was supported by Brazilian CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior). L. F. Marins is a research fellow from CNPq (Proc. No. 304675/2011-3).
References Alavi, S.M.H., Cosson, J., 2006. Sperm motility in fishes. (II) Effects of ions and osmolality: a review. Cell Biol. Int. 30, 1–14. Almeida, D.V., Martins, C.M.G., Figueiredo, M.A., Lanes, C.F.C., Bianchini, A., Marins, L.F., 2013. Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio). Transgenic Res. 22 (1), 75–88. Bencharif, D., Amirat, L., Pascal, O., Anton, M., Schmitt, E., Desherces, S., Delhomme, G., Langlois, M.L., Barriere, P., Larrat, M., Tainturier, D., 2010. The advantages of combining low-density lipoproteins with glutamine for cryopreservation of canine semen. Reprod. Domest. Anim. 45 (2), 189–200. Bessey, C., Devlin, R.H., Liley, N.R., Biagi, C.A., 2004. Reproductive performance of growth-enhanced transgenic coho salmon (Oncorhynchus kisutch). Trans. Am. Fish. Soc. 133, 1205–1220. Bobe, J., Labbé, C., 2010. Egg and sperm quality in fish. Gen. Comp. Endocrinol. 165, 535–548. Boe-Hansen, G.B., Christensen, P., Vibjerg, D., Nielsen, M.B.F., Hedeboe, A.M., 2008. Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology 69, 728–736. Bragadin, M., Cima, F., Ballarin, L., Manente, S., 2006. Irgarol inhibits the synthesis of ATP in mitochondria from rat liver. Chemosphere 64, 1898–1903. Carson, F.L., Hladik, C., 2009. Histotechnology—A Self-instructional Text, 3rd ed. ASCP Press, Chicago. Cook, J.T., McNiven, M.A., Richardson, G.F., Sutterlin, A.M., 2000. Growth rate, body composition and feed digestibility/conversion of growthenhanced transgenic Atlantic salmon (Salmo salar). Aquaculture 188, 15–32. Devlin, R.H., Yesaki, T.Y., Biagi, C.A., Donaldson, E.M., Swanson, P., Chan, W.K., 1994. Extraordinary salmon growth. Nature 371, 209–210. Devlin, R.H., Sundström, L.F., Muir, W.M., 2006. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends Biotechnol. 24, 89–97. Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Marins, L.F., 2007a. Improving the production of transgenic fish germline: in vivo mosaicism evaluation by GFP transgene co-injection strategy. Genet. Mol. Biol. 30, 31–36. Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Proietti, M.C., Marins, The effect of GH overexpression on GHR and L.F., 2007b. IGF-I gene regulation in different genotypes of GH transgenic zebrafish. Comp. Biochem. Physiol. Part D: Genomics Proteomics 2, 228–233. Fitzpatrick, J.L., Akbarashandiz, H., Sakhrani, D., Biagi, C.A., Pitcher, T.E., Devlin, R.H., 2011. Cultured growth hormone transgenic salmon are reproductively out-competed by wild-reared salmon in semi-natural mating arenas. Aquaculture 312, 185–191. Gomez, J.M., Boujard, T., Fostier, A., Le Bail, P.Y., 1996. Characterization of growth hormone nycthemeral plasma profiles in catheterized rainbow trout (Oncorhynchus mykiss). J. Exp. Zool. 274, 171–180. Harrison, R.A.P., Vickers, S.E., 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88, 343–352. He, S., Woods, L.C., 2004. Efects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membrane and mitochondria to striped bass (Morone saxatilis) sperm. Cryobiology 48, 254–262. Herbert, N.A., Armstrong, J.D., Björnsson, B.T., 2001. Evidence that growth hormone-induced elevation in rotine metabolism of juvenile Atlantic salmon is a result of increased spontaneous activity. J. Fish Biol. 59, 754–757.
M.A. Figueiredo et al. / Animal Reproduction Science 139 (2013) 162–167 Jansson, J.O., Edén, S., Isaksson, O., 1985. Sexual dimorphism in the control of growth hormone secretion. Endocr. Rev. 6, 128–150. Kanuga, M.K., Benner, M.J., Doble, J.A., Wilson-Leedy, J.G., Robison, B.D., Ingermann, R.L., 2011. Effect of aging on male reproduction in zebrafish (Danio rerio). J. Exp. Zool. 315, 156–161. Kuradomi, R.Y., Figueiredo, M.A., Lanes, C.F.C., Rosa, C.E., Almeida, D.V., Maggioni, R., Silva, M.D.P., Marins, L.F., 2011. GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model. Transgenic Res. 20, 513–521. McKenzie, D.J., Martinez, I.R., Morales, A., Acosta, J., Taylor, E.W., Steffensen, J.F., Estrada, M.P., 2000. Metabolic rate, exercise performance and hypoxia tolerance of growth hormone transgenic tilapia (Oreochromis sp.). Comp. Biochem. Physiol. 126, S66. McKenzie, D.J., Martínez, R., Morales, A., Acosta, J., Morales, R., Taylor, E.W., Steffensen, J.F., Estrada, M.P., 2003. Effects of growth hormone transgenesis on metabolic rate, exercise performance and hypoxia tolerance in tilapia hybrids. J. Fish Biol. 63, 398–409. Moreau, D.T.R., Conway, C., Fleming, I.A., 2011. Reproductive performance of alternative male phenotypes of growth hormone transgenic Atlantic salmon (Salmo salar). Evol. Appl. 4, 736–748. Mori, T., Hiraka, I., Kurata, Y., Kawachi, H., Mano, N., Devlin, R.H., Nagoya, H., Araki, K., 2007. Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GHtransgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity. Gen. Comp. Endocrinol. 151, 42–54. Muir, W.M., Howard, R.D., 1999. Possible ecological risks of transgenic organism release when transgenes affect mating success: sexual selection and the Trojan gene hypothesis. Proc. Natl. Acad. Sci. U.S.A. 96, 13853–13856. Nam, Y.K., Noh, J.K., Cho, Y.S., Cho, H.J., Cho, K.N., Kim, C.G., Kim, D.S., 2001. Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Res. 10, 353–362.
167
Pursel, V.G., Johnson, L.A., 1975. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99–102. Rosa, C.E., Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Monserrat, J.M., Marins, L.F., 2008. Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 149, 209–214. Rosa, C.E., Kuradomi, R.Y., Almeida, D.V., Lanes, C.F.C., Figueiredo, M.A., Dytz, A.G., Fonseca, D.B., Marins, L.F., 2010. GH overexpression modifies muscle expression of anti-oxidant enzymes and increases spinal curvature of old zebrafish. Exp. Gerontol. 45, 449–456. Rosa, C.E., Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Marins, L.F., 2011. Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model. Transgenic Res. 20, 85–89. Schulz, R.W., de Franc¸a, L.R., Lareyre, J.J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165 (3), 390–411. Sies, H., Jones, D.P., 2007. Oxidative stress. In: Fink, G. (Ed.), Encyclopaedia of Stress. Elsevier, San Diego, pp. 45–48. Statistix, 2008. Statistix 9 for Windows. Analytical Software. Studzinski, A.L., Almeida, D.V., Lanes, C.F., Figueiredo, M.A., Marins, L.F., 2009. SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio). Gen. Comp. Endocrinol. 161, 67–72. Varela Junior, A.S., Corcini, C.D., Gheller, S.M.M., Jardim, R.D., Lucia Jr., T., Streit Jr., D.P., Figueiredo, M.R.C., 2012. Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum. Theriogenology 78, 244–251. Zhang, W.M., Lin, H.R., Peter, R.E., 1994. Episodic growth hormone secretion in the grass carp, Ctenopharyngodon idellus. Gen. Comp. Endocrinol. 95, 337–341. Zini, A., Kamal, K., Phang, D., Willis, J., Jarvi, K., 2001. Biologic variability of sperm DNA denaturation in infertile men. Urology 58, 258–261.
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males Marcio A. Figueiredo a , Raíssa V. Fernandes a , Ana L. Studzinski a , Carlos E. Rosa a , Carine D. Corcini b , Antônio S. Varela Junior a , Luis F. Marins a,∗ a b
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas – UFPEL, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 4 August 2012 Received in revised form 18 March 2013 Accepted 26 March 2013 Available online 4 April 2013 Keywords: Growth hormone Transgenesis Reproduction Zebrafish Trojan effect
a b s t r a c t Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growth hormone is a polypeptide produced and secreted by the adenohypophysis, promoting, as a main effect, somatic growth in vertebrates. Although the manipulation of GH gene by transgenesis have been showing promising results regarding fish growth (Devlin et al., 1994; Nam et al., 2001), its excess may lead to unwanted collateral effects. GH is well known by its role in many processes besides growth, having pleiotropic effects over morphology, physiology, metabolism, immunology, reproduction
∗ Corresponding author at: Universidade Federal do Rio Grande – FURG, Instituto de Ciências Biológicas, Av. Itália, Km 8, 96203-900 Rio Grande, RS, Brazil. Tel.: +55 53 32935191. E-mail address: [email protected] (L.F. Marins). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.03.012
and behavior (Devlin et al., 2006). Therefore, it is obvious that the maintenance of this hormone under supraphysiological levels may lead to a series of undesirable effects over other systems within the organism. Mori et al. (2007) have observed an alteration in the expression of genes related to immune and reproductive processes in amago salmon (Oncorhynchus masou). There were also observed a significant increase of metabolic rates and oxygen consumption in Atlantic salmon (Cook et al., 2000; Herbert et al., 2001) and tilapias (McKenzie et al., 2000, 2003), as a result of GH superexpression or its administration. In addition, GH-transgenesis in coho salmon decreases sperm quality in comparison to wild males (Fitzpatrick et al., 2011), and affects reproductive behavior during competition with hatchery males (Bessey et al., 2004). In the same way, Moreau et al. (2011) showed that GH-transgenic Atlantic salmon had lower overall fertilization success and
M.A. Figueiredo et al. / Animal Reproduction Science 139 (2013) 162–167
reduced breeding performance relative to nontransgenics. Aiming to study the collateral effects caused by GH excess, our research group has developed a transgenic zebrafish (Danio rerio) model that overexpresses GH from the marine silverside fish Odontestes argentinensis (Figueiredo et al., 2007a). This lineage, named F0104, has been previously studied in terms of growth and gene expression from the somatotropic axis (Figueiredo et al., 2007b), metabolic rate, reactive oxygen species (ROS) production and aging (Rosa et al., 2008, 2010, 2011), intracellular signaling (Studzinski et al., 2009), muscular structure (Kuradomi et al., 2011) and salinity tolerance (Almeida et al., 2013). However, this model remains without an analysis concerning the reproductive parameters. Regarding reproduction, GH transgenesis may have a sex-dependant effect, once a sex-specific pulsatile secretion pattern of this hormone is already well characterized in mammals. In this vertebrate group, females present more frequent and less pronounced secretion peaks along the day, while males exhibit peaks, although less frequent, with larger amplitude (Jansson et al., 1985). Despite little information is available in fish, there is evidence that GH may also be secreted in an episodic fashion in carp (Zhang et al., 1994) and in a sex-dependant way in the rainbow trout (Gomez et al., 1996). Regarding transgenic zebrafish model, the object from the present study, the constant GH overexpression along time may induce a profile similar to that observed in females, thus leading to more pronounced effect in males. Considering that GH may interfere in the sexual maturation process and that transgenesis can alter the natural sex-dependant expression feature of this hormone, the present study aimed to evaluate the reproductive parameters of transgenic adult zebrafish males from F0104 lineage. Two-years-old fish were used since it has been demonstrated that at this age, even at the end of reproductive cycle, sperm quality did not differ between young and old zebrafish (Kanuga et al., 2011). Therefore, the spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality and DNA integrity, comparing transgenic and non-transgenic fish. In addition, histological slides were made aiming to verify if transgenesis caused any effect over the tissue’s structure or the presence of characteristic cell types.
2. Materials and methods 2.1. Zebrafish Transgenic and non-transgenic zebrafish from lineage F0104 were kept in a closed system of water circulation containing 50 aquaria, with 15 l of volume each, oxygen levels near saturation, pH close to 7, photoperiod 14L:10D and temperature of 28 ◦ C. Animals were fed ad libitum, twice a day with a commercial fish food (Tetra ColorBits) enriched with alive nauplii of Artemia sp.
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2.2. Testes excision Fish at the same age (two years-old ± one month) were used in the procedures. The pair of testes, from 11 transgenic hemizygous males and 14 non-transgenic (control group), were excised and immediately placed in 100 L of Beltsville Thawing Solution (BTS) (Pursel and Johnson, 1975). Bathing in the aforementioned solution allows the collection of spermatozoa, which were not encysted, and keeps the tissue intact for further histological analyses. Following the bath, each gonad was fixed in buffered 4% formaldehyde solution during 24 h for the histological analyses. 2.3. Motility and the motility period of sperm Sperm motility and the motility period were evaluated in a microscope of phase-contrast using 1 L of zebrafish semen in BTS and 99 L of distilled water (25 ◦ C), intending to activate the spermatozoa. The motility period comprised, in seconds, the stage between the spermatozoa activation and the absence of progressive movement (a straight line movement). 2.4. Sperm membrane integrity Aiming to analyze the cytoplasmic membrane integrity of spermatozoa, two probes were used to allow differentiation between the two cellular types: intact and damaged. For evaluation, 5 L sperm sample was diluted in 20 L of working isotonic saline solution, including: 1.7 mM formaldehyde, 20 M carboxyfluorescein diacetate (CFDA), and 7.3 M propidium iodide (PI) (Varela Junior et al., 2012). Two hundred spermatozoa were analyzed and those presenting an intact membrane went through a hydrolysis process, transforming the CFDA into carboxyfluorescein, which accumulates in all compartments emitting green fluorescence as a singular characteristic. Spermatozoa presenting damaged membrane do not accumulate CFDA. In addition, damaged spermatozoa incorporate PI emitting a red fluorescence (Harrison and Vickers, 1990). 2.5. Mitochondrial functionality Intending to evaluate the mitochondrial sperm functionality, rhodamine probe 123 was used (Rh 123 – Sigma, Brazil). Sperm was evaluated after incubation of a 5 L sample with a 20 L Rh 123 solution (13 M) at 20 ◦ C for 10 min. Sperm with positive rhodamine staining (green fluorescence) were considered as having functional mitochondria. Conversely, nonfunctional mitochondria were characterized by negative rhodamine staining (sperm with no fluorescence). The rate of mitochondrial functionality was determined by the proportion of sperm emitting green fluorescence compared with total sperm (He and Woods, 2004). 2.6. DNA integrity Sperm DNA integrity was evaluated after putting a 10 L sperm sample in 10 L TNE (0.01 M Tris–HCl; 0.15 M NaCl;
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0.001 M EDTA; pH 7.2). After 30 s, 40 L of Triton solution 1× was added and, 30 s later, 10 L of acridine orange was added (2 mg/mL in deionized H2 O). The evaluation was done after 5 min, without exceeding 1 min of slide exposure. Sperm with green fluorescence were considered as having intact DNA, whereas those with red or orange fluorescence were considered as having denatured DNA (Bencharif et al., 2010). The frequency of DNA integrity was determined by the proportion of sperm emitting green fluorescence compared with the total number of sperm (green, red, or orange fluorescence) (Varela Junior et al., 2012).
3. Results
2.7. Histology
3.2. Mitochondrial functionality
Sexual maturation was examined by analysis of histological slides from testes. After 24 h immersed in buffered paraformaldehyde, the gonads were dehydrated by alcohol baths, with growing concentrations, followed by the processes of diaphanisation (xylol), impregnation and inclusion in Paraplast Xtra (P3808 – Sigma, Brazil), according to Carson and Hladik (2009). After inclusion, the material was sliced in a motorized rotary microtome (Leica RM2255) with thickness of 6 m. The fixed slides were stained with hematoxylin and eosin (Carson and Hladik, 2009). After the staining process, the material was visualized in an optic microscope under 100× magnification. Sexually mature testes presented the four cellular stages: spermatogonium, spermatid, spermatocyte and spermatozoid.
The spermatozoa from transgenic fish have presented a mean mitochondrial functionality of 51.1% (±5.4). The mean mitochondrial functionality of non-transgenic animals was 89.6% (±3.6), which was significantly higher than the observed in transgenic zebrafish (p < 0.05) (Fig. 1C).
3.1. Motility and motility period of spermatozoa Non-transgenic fish presented mean spermatic motility of 91.8% (±2.9), which was significantly higher (p < 0.05) than in transgenic individuals (36.4 ± 12.4%) (Fig. 1A). After verifying the percentage of motility, the motility period time was counted in seconds. The mean time observed until ceasing progressive movement of non-transgenic fish was 103.6 s (±12.9), which was significantly higher (p < 0.05) to mean motility period of transgenic fish (41.4 ± 12.7 s) (Fig. 1B).
3.3. Sperm membrane integrity Concerning the group of transgenic animals, the mean of fish presenting spermatozoa with intact membrane was 52.7% (±4.6). On the other hand, a significantly higher mean of 91.3% (±1.8) was found for non-transgenic fish presenting spermatozoa with intact membrane (p < 0.05) (Fig. 1D). 3.4. DNA integrity
2.8. Fertilization and hatching frequency Aiming to evaluate reproductive parameters, a breeding assessment was performed between non-transgenic couples (n = 5) and a crossbreeding between transgenic males and non-transgenic females (n = 5). Different females were used for each male. The eggs were collected in traps 1 h after spawning, intending to examine the fertilization rate in a stereoscopic microscope. Eggs characterized by 4–8 blastomeres were considered fertilized. Subsequently, all eggs were counted and incubated at 28 ◦ C until hatching. The hatching rate was determined 3 days after spawning and expressed as the percentage of hatched larvae in relation to total number of incubated eggs.
2.9. Statistical analyses The statistical analyses of GH transgenic and nontransgenic fish groups were realized using the software Statistix 9.0, Analytical Software (Tallahassee, FL, USA) (Statistix, 2008). Shapiro–Wilk test was run for analysis of the samples normality. The parameters considered with normal distribution were submitted to variance analysis, comparing transgenic and non-transgenic animals, followed by Tukey’s test HSD with significance level of 5%. The parameters without normal distribution were submitted to Kruskal–Wallis nonparametric test with significance level of 5%.
The group of transgenic fish presented significantly lower DNA integrity (p < 0.05) when compared with non-transgenic animals. The mean DNA integrity of spermatozoa from non-transgenic fish was 81.4% (±4.1), while the observed for transgenic fish was only 33.3% (±6.3) (Fig. 1E). 3.5. Fertilization and hatching frequency The results from fertilization and hatching are shown in Fig. 1F. The fertilization rate observed in non-transgenic fish was 90% (±1.4), while this parameter was significantly lower in transgenic individuals (76 ± 3.2%) (p < 0.05). The same tendency was observed concerning the hatching frequency. The percentage of hatched eggs from nontransgenic animals was 85% (±9.1) and from transgenic fish was 42% (±6.9) (p < 0.05). 4. Discussion The present study has shown that GH gene transgenesis produced a negative effect over the reproductive success in two-years-old zebrafish males from lineage F0104. All the analyzed parameters presented a significant decrease of sperm quality resulting in important consequences on the fertilization and spawning viability. These results may have important repercussions concerning the viability of the mentioned technology when used intending to increase
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Fig. 1. Comparison of spermatic parameters between GH-transgenic (T) and non-transgenic (NT) zebrafish males. (A) % of motile cells; (B) motility period (s); (C) % of functional mitochondria; (D) % membrane integrity; (E) % DNA integrity; (F) % fertilization and hatching. Asterisks represent statistically significant differences (p < 0.05).
production in aquaculture, as the amount of offspring generated by transgenic mates with such reproductive success could be a limiting factor. Besides, transgenic fish presenting decreased reproductive viability could represent a great environmental risk according to Trojan gene hypothesis, which postulates that a transgenic fish with advantage in sexual selection, associated with minor spawning viability, could extinguish an entire population of wild animals after a few generations (Muir and Howard, 1999). The first parameters analyzed in the present study were motility and motility period of sperm. Motility refers to spermatozoid capacity of moving toward the oocyte and it is a crucial factor for fertilization. Also, motility is activated by changes in intracellular ion concentrations, which are dependant of ATPases activity. These enzymes can alter the cellular ionic composition and produce a membrane hyperpolarization, inducing sperm motility (Schulz et al., 2010). ATPases are highly dependent on ATP and the
mitochondria play an important role within this mechanism (Bragadin et al., 2006). In the same sense, motility period is considered as the time in which the sperm keeps on movement and depends on the available energy. The obtained results concerning these two parameters indicate that spermatozoa from GH-transgenic fish present a significant diminishing of 60% approximately in relation to non-transgenic animals (Fig. 1A and B). These results are supported by the significant percentage decrease in functional mitochondria of 43% observed in transgenic individuals (Fig. 1C). Therefore, it is hypothesized that transgenic males possess a lower amount of available ATP, turning them less viable to fertilization. One possible explanation for decreased mitochondrial functionality could be the increased production of reactive oxygen species (ROS) induced by the excess of GH. Formerly, it was demonstrated that fish from lineage F0104 exhibit a significant increase in the metabolic rate and ROS generation in
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muscle, and decreased expression of genes from the antioxidant defense system (ADS) in liver and muscle (Rosa et al., 2008, 2011). The present scenario, with increased ROS associated with decreased ADS from testes, could represent an oxidative stress situation in which could cause great damages in the mitochondrial membrane and in its functionality (Sies and Jones, 2007). Thus, the spermatogonia exposed to elevated amount of ROS could be originating sperm with lower quantity of functional mitochondria and affecting of motility and motility period. The same way that elevated amount of ROS may alter the mitochondrial membrane, the spermatozoid membrane can undergo the same effect. The obtained result regarding the sperm membrane integrity has shown a significant diminishing of approximately 42% in transgenic fish (Fig. 1D). This decreasing may have an important effect over spermatozoa motility, once the membrane guides all water and ion exchanges between intra and extracellular environments and triggers the flagella movements (Alavi and Cosson, 2006). The plasmatic membrane of spermatozoa from teleost fish is a key component in gamete fusion, due the absence of acrosome in this vertebrate group, which directly affects fertilization (Bobe and Labbé, 2010). Besides membranes, ROS can also target the DNA molecule. Under a situation of oxidative stress, DNA may suffer strands damage, base removal or nucleotide modification, particularly in guanine rich regions. When these alterations are not repaired, they can cause serious damage to the cell and compromise its viability. In the present study, there was observed a significant reduction of approximately 60% in DNA integrity from transgenic animals (Fig. 1E). Although the DNA quality does not affects the spermatozoid’s fertilization capacity, this parameter is crucial for the embryo development (Zini et al., 2001; Boe-Hansen et al., 2008). Considering that all analyzed parameters indicate a significant diminishing of sperm quality from transgenic fish, a direct consequence over fertilization and hatching rates is expected. Fig. 1F summarizes the results of fertilization and hatching rates evincing significant decreases of 16 and 51%, respectively, even though the histological analysis have detected all characteristic cell types of mature testes in transgenic fish. These are concerning results when considering GH transgenesis application in aquaculture, if transgenic mates with such reproductive deficiency degree are not viable. These results are also concerning under an environmental point of view. Muir and Howard (1999) have developed a mathematic model that predicts extinction, after few generations, of wild populations in which transgenic fish presenting mating advantage associated with diminished viability of its offspring were introduced. However, this hypothesis is based on models built on data obtained under controlled laboratory conditions. Actually, within a variable environment, with low food availability and high competition at different levels, it is presumable that the transgenic animal will not achieve the same reproductive success from the wild-type and will be unable to spread its genes easily. Aiming to clarify this question, research involving reproductive behavior of transgenic individuals is made necessary to verify the existence of a possible
sexual selection advantage in relation to non-transgenic fish.
Acknowledgements This work was supported by Brazilian CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior). L. F. Marins is a research fellow from CNPq (Proc. No. 304675/2011-3).
References Alavi, S.M.H., Cosson, J., 2006. Sperm motility in fishes. (II) Effects of ions and osmolality: a review. Cell Biol. Int. 30, 1–14. Almeida, D.V., Martins, C.M.G., Figueiredo, M.A., Lanes, C.F.C., Bianchini, A., Marins, L.F., 2013. Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio). Transgenic Res. 22 (1), 75–88. Bencharif, D., Amirat, L., Pascal, O., Anton, M., Schmitt, E., Desherces, S., Delhomme, G., Langlois, M.L., Barriere, P., Larrat, M., Tainturier, D., 2010. The advantages of combining low-density lipoproteins with glutamine for cryopreservation of canine semen. Reprod. Domest. Anim. 45 (2), 189–200. Bessey, C., Devlin, R.H., Liley, N.R., Biagi, C.A., 2004. Reproductive performance of growth-enhanced transgenic coho salmon (Oncorhynchus kisutch). Trans. Am. Fish. Soc. 133, 1205–1220. Bobe, J., Labbé, C., 2010. Egg and sperm quality in fish. Gen. Comp. Endocrinol. 165, 535–548. Boe-Hansen, G.B., Christensen, P., Vibjerg, D., Nielsen, M.B.F., Hedeboe, A.M., 2008. Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology 69, 728–736. Bragadin, M., Cima, F., Ballarin, L., Manente, S., 2006. Irgarol inhibits the synthesis of ATP in mitochondria from rat liver. Chemosphere 64, 1898–1903. Carson, F.L., Hladik, C., 2009. Histotechnology—A Self-instructional Text, 3rd ed. ASCP Press, Chicago. Cook, J.T., McNiven, M.A., Richardson, G.F., Sutterlin, A.M., 2000. Growth rate, body composition and feed digestibility/conversion of growthenhanced transgenic Atlantic salmon (Salmo salar). Aquaculture 188, 15–32. Devlin, R.H., Yesaki, T.Y., Biagi, C.A., Donaldson, E.M., Swanson, P., Chan, W.K., 1994. Extraordinary salmon growth. Nature 371, 209–210. Devlin, R.H., Sundström, L.F., Muir, W.M., 2006. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends Biotechnol. 24, 89–97. Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Marins, L.F., 2007a. Improving the production of transgenic fish germline: in vivo mosaicism evaluation by GFP transgene co-injection strategy. Genet. Mol. Biol. 30, 31–36. Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Proietti, M.C., Marins, The effect of GH overexpression on GHR and L.F., 2007b. IGF-I gene regulation in different genotypes of GH transgenic zebrafish. Comp. Biochem. Physiol. Part D: Genomics Proteomics 2, 228–233. Fitzpatrick, J.L., Akbarashandiz, H., Sakhrani, D., Biagi, C.A., Pitcher, T.E., Devlin, R.H., 2011. Cultured growth hormone transgenic salmon are reproductively out-competed by wild-reared salmon in semi-natural mating arenas. Aquaculture 312, 185–191. Gomez, J.M., Boujard, T., Fostier, A., Le Bail, P.Y., 1996. Characterization of growth hormone nycthemeral plasma profiles in catheterized rainbow trout (Oncorhynchus mykiss). J. Exp. Zool. 274, 171–180. Harrison, R.A.P., Vickers, S.E., 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88, 343–352. He, S., Woods, L.C., 2004. Efects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membrane and mitochondria to striped bass (Morone saxatilis) sperm. Cryobiology 48, 254–262. Herbert, N.A., Armstrong, J.D., Björnsson, B.T., 2001. Evidence that growth hormone-induced elevation in rotine metabolism of juvenile Atlantic salmon is a result of increased spontaneous activity. J. Fish Biol. 59, 754–757.
M.A. Figueiredo et al. / Animal Reproduction Science 139 (2013) 162–167 Jansson, J.O., Edén, S., Isaksson, O., 1985. Sexual dimorphism in the control of growth hormone secretion. Endocr. Rev. 6, 128–150. Kanuga, M.K., Benner, M.J., Doble, J.A., Wilson-Leedy, J.G., Robison, B.D., Ingermann, R.L., 2011. Effect of aging on male reproduction in zebrafish (Danio rerio). J. Exp. Zool. 315, 156–161. Kuradomi, R.Y., Figueiredo, M.A., Lanes, C.F.C., Rosa, C.E., Almeida, D.V., Maggioni, R., Silva, M.D.P., Marins, L.F., 2011. GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model. Transgenic Res. 20, 513–521. McKenzie, D.J., Martinez, I.R., Morales, A., Acosta, J., Taylor, E.W., Steffensen, J.F., Estrada, M.P., 2000. Metabolic rate, exercise performance and hypoxia tolerance of growth hormone transgenic tilapia (Oreochromis sp.). Comp. Biochem. Physiol. 126, S66. McKenzie, D.J., Martínez, R., Morales, A., Acosta, J., Morales, R., Taylor, E.W., Steffensen, J.F., Estrada, M.P., 2003. Effects of growth hormone transgenesis on metabolic rate, exercise performance and hypoxia tolerance in tilapia hybrids. J. Fish Biol. 63, 398–409. Moreau, D.T.R., Conway, C., Fleming, I.A., 2011. Reproductive performance of alternative male phenotypes of growth hormone transgenic Atlantic salmon (Salmo salar). Evol. Appl. 4, 736–748. Mori, T., Hiraka, I., Kurata, Y., Kawachi, H., Mano, N., Devlin, R.H., Nagoya, H., Araki, K., 2007. Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GHtransgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity. Gen. Comp. Endocrinol. 151, 42–54. Muir, W.M., Howard, R.D., 1999. Possible ecological risks of transgenic organism release when transgenes affect mating success: sexual selection and the Trojan gene hypothesis. Proc. Natl. Acad. Sci. U.S.A. 96, 13853–13856. Nam, Y.K., Noh, J.K., Cho, Y.S., Cho, H.J., Cho, K.N., Kim, C.G., Kim, D.S., 2001. Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Res. 10, 353–362.
167
Pursel, V.G., Johnson, L.A., 1975. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99–102. Rosa, C.E., Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Monserrat, J.M., Marins, L.F., 2008. Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 149, 209–214. Rosa, C.E., Kuradomi, R.Y., Almeida, D.V., Lanes, C.F.C., Figueiredo, M.A., Dytz, A.G., Fonseca, D.B., Marins, L.F., 2010. GH overexpression modifies muscle expression of anti-oxidant enzymes and increases spinal curvature of old zebrafish. Exp. Gerontol. 45, 449–456. Rosa, C.E., Figueiredo, M.A., Lanes, C.F.C., Almeida, D.V., Marins, L.F., 2011. Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model. Transgenic Res. 20, 85–89. Schulz, R.W., de Franc¸a, L.R., Lareyre, J.J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165 (3), 390–411. Sies, H., Jones, D.P., 2007. Oxidative stress. In: Fink, G. (Ed.), Encyclopaedia of Stress. Elsevier, San Diego, pp. 45–48. Statistix, 2008. Statistix 9 for Windows. Analytical Software. Studzinski, A.L., Almeida, D.V., Lanes, C.F., Figueiredo, M.A., Marins, L.F., 2009. SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio). Gen. Comp. Endocrinol. 161, 67–72. Varela Junior, A.S., Corcini, C.D., Gheller, S.M.M., Jardim, R.D., Lucia Jr., T., Streit Jr., D.P., Figueiredo, M.R.C., 2012. Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum. Theriogenology 78, 244–251. Zhang, W.M., Lin, H.R., Peter, R.E., 1994. Episodic growth hormone secretion in the grass carp, Ctenopharyngodon idellus. Gen. Comp. Endocrinol. 95, 337–341. Zini, A., Kamal, K., Phang, D., Willis, J., Jarvi, K., 2001. Biologic variability of sperm DNA denaturation in infertile men. Urology 58, 258–261.