mRNA expression of GnRH variants and receptors in the brain, pituitary and ovaries of pejerrey
(Odontesthes bonariensis) in relation to the reproductive status
L. G. GuilgurÆC. A. Stru¨ssmann ÆG. M. Somoza
Received: 4 February 2008 / Accepted: 20 March 2008 / Published online: 17 April 2008 ÓSpringer Science+Business Media B.V. 2008
Abstract The present study examined the differen- tial mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition,
suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.
Keywords mRNA levelsGnRH GnRH receptorsOvary Reproduction
Introduction
Gonadotropin-releasing hormone (GnRH) is synthesized in the brain and acts on GnRH receptors (GnRH-Rs) in the anterior pituitary to stimulate the synthesis and release of gonadotropins (GtHs) in vertebrates (Millar et al. 2004). It is also known that many vertebrate species express more than one GnRH variant in their brains (Kah et al. 2007). Then it can be difficult to understand the role of GnRH in reproduction because the analysis must take into consideration the functional significance of these multiple GnRH forms. This prob- lem is particularly complex in those teleost species which express three GnRHs in the brain (Somoza et al.
2002a; Lethimonier et al. 2004; Millar et al. 2004;
Guilgur et al. 2006). Furthermore, the existence of multiple GnRH forms and GnRH receptor types, together with their anatomical location, suggests that a complex interplay occurs between ligands and receptors not only on the regulation of pituitary hormone secre- tion but also on other still unknown physiological functions (Lethimonier et al. 2004; Moncaut et al.
2005; Gonza´lez-Martı´nez et al. 2006; Guilgur et al.
2006). In this regard, it is important to note that, except L. G. GuilgurG. M. Somoza (&)
Laboratorio de Ictiofisiologı´a y Acuicultura, Instituto de Investigaciones Biotecnolo´gicas-Instituto Tecnolo´gico Chascomu´s (IIB-INTECH), cc 164 (B7130IWA) Chascomu´s, Buenos Aires, Argentina
e-mail: [email protected] C. A. Stru¨ssmann
Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-Ku, Tokyo, Japan
DOI 10.1007/s10695-008-9215-4
for the hypophysiotropic function of the preoptic- hypothalamic GnRH form (also named GnRH-I), the physiological roles of other GnRHs are poorly known.
The examination of the expression of the various GnRH and GnRH-R genes in the brain and pituitary gland might help to understand the function of each of the variants in the reproductive process. However, information on the abundance of GnRH and GnRH-R mRNA is greatly limited, especially in teleost species having three molecular forms of GnRH in their brain (Okuzawa et al.2003). Also, the expression of these genes is not limited to the central nervous system.
Indeed, the presence of GnRH peptides has been demonstrated in fish gonads (Nabissi et al.1997; Pati and Habibi 1998; Von Schalburg et al.1999). Also, the expression of GnRH mRNA in the gonads was found to be maturation stage-dependent, supporting a potential autocrine/paracrine regulatory function of GnRH during gametogenesis (Von Schalburg and Sherwood 1999a, b; Uzbekova et al. 2001, 2002).
However, information concerning GnRHs and GnRH-Rs gene expression in teleost gonads is still fragmentary (Madigou et al.2002).
Our experimental model, the pejerrey (Odontes- thes bonariensis), is a multiple spawner fish species from the inland waters of Argentina. It is a commer- cially important species and also a very popular game fish (Lo´pez et al. 2001). The biological basis of its reproductive cycle and the histological characteristics of the process of gonadal maturation in wild and captive-reared pejerrey have been fairly well described (Calvo and Morriconi 1972; Stru¨ssmann 1989; Fernandino et al. 2006; Miranda et al. 2007).
Pejerrey expresses three distinct forms of GnRH, namely salmon GnRH (sGnRH, GnRH-III), chicken GnRH-II (cGnRH-II, GnRH-II) and pejerrey GnRH (pjGnRH or mdGnRH, GnRH-I) in the brain. These forms have been characterized using both biochem- ical and molecular techniques (Montaner et al.2001;
Guilgur et al.2007), and they have also been shown to exhibit a differential neuroanatomical distribution (Stefano et al. 2000; Miranda et al. 2003). Thus, pjGnRH is the only variant detected in the pituitary gland, suggesting a hypophysiotropic function (Stef- ano et al. 1997; Somoza et al. 2002b). In addition, full-length cDNA sequences encoding for two GnRH-Rs (pjGnRH-R I and pjGnRH-R II) have been characterized in this species (GenBank accession numbers DQ875596 and DQ875595, respectively).
In this context, the aim of the present study was to investigate the mRNA expression levels of sGnRH, cGnRH-II, pjGnRH, and pjGnRH receptors I and II in the brain, pituitary gland and ovary of female pejerrey during oogenesis, in order to obtain funda- mental information to understand the roles of these three hormones and their receptors in the reproduc- tion of this species.
Materials and methods
Source of the animals and sample collection The fish were obtained from a stock maintained under natural environmental conditions in outdoor ponds at the IIB-INTECH rearing facilities. The sampling points were a priori selected according the knowledge of the reproductive cycle of this species following Calvo and Morriconi (1972) and Stru¨ssmann (1989): May (middle of autumn, early gonadal development), August (end of winter, final maturation) and November (late spring, post spawning). The fish used were 2-year-old adult fish and were sampled in May, August and November 2005.
Before tissue collection, fish were first anesthetized with benzocaine and their body weight and standard length were recorded. Then, they were sacrificed by rapid decapitation in accordance with the UFAW Handbook on the Care and Management of Laboratory Animals (http://www.ufaw.org.uk/pubs.htm#Lab) and local regulations. Brains and pituitaries were quickly excised and stored in RNAlater (SIGMAÒ) at –80°C until use.
Next, the gonads were excised and their weight recorded to calculate the gonadosomatic index (GSI%, 1009 gonadal weight/body weight). One gonad was fixed in Bouin’s solution for histological observation of game- togenesis and sexual maturation and the contra-lateral one was processed as for the brains and pituitaries for molecular analysis.
Bouin-fixed gonads were processed by routine techniques (embedding, sectioning, and staining with hematoxylin and eosin) for the obtention of represen- tative histological sections. Prior to the molecular analysis, the sections of all experimental animals were observed under a microscope and all samples taken in May were classified asresting, the ones taken in August as developing and those from November as spent following criteria of Stru¨ssmann (1989). Thus, the different groups were: (1) May,resting stage (ovaries
with a thick ovarian tunica and containing only primary growth, previtellogenic oocytes up to and including cortical alveoli oocytes but no vitellogenic oocytes;
mean GSI%=1.08±0.22,n=6), (2) August,devel- oping stage (ovaries with oocytes having abundant cortical alveoli and a centrally located germinal vesicle;
mean GSI%=2.28±0.14, n=6), and (3) Novem- ber, spent stage(ovaries with a moderately distended ovarian tunica and containing mostly pre-vitellogenic oocytes; early vitellogenic oocytes, if present, are few and show signs of atresia; may or not contain also post- ovulatory follicles and or atretic bodies; mean GSI%=1.522±0.25,n=6).
GnRH and GnRH-R mRNA expression and data analyses
Total RNA was extracted from brains, pituitaries, and gonads using Trizol reagent (InvitrogenTM; Life Technologies) following the manufacturer’s instruc- tions. Real time PCR was performed for each GnRH and GnRH-R forms and pejerrey b-actin using samples from six females for each of the reproductive stages characterized above. For this purpose, RNA samples were treated with DNase I and reverse- transcribed using SuperScript III RNase H-(Invitro- genTM, Life Technologies) and oligo(dT)12–18. The primers used (pjGnRHFw-pjGnRHRv, cGnRH-IIFw- cGnRH-IIRv, sGnRHFw-sGnRHRv, pjGnRH-RIFw- pjGnRH-RIRv, pjGnRH-RIIFw-pjGnRH-RIIRv and bactinFw-bactinRv were designed using the Primer Express Software (Applied Biosystems) and are detailed in Table1.
Real time PCRs were performed in a 20-ll reaction volume containing 10ll of 2X SYBRÒ Premix Ex TaqTM (TaKaRa), 1 ll of first strand cDNA (approximately 25 ng as measured by spec- trophotometry at 260 nm), and 5 pmol of each primer using a ABI PRISM 7300 thermocycler (Applied Biosystems). Thermal cycling conditions consisted in an initial step of 10 s at 95°C followed by 40 cycles of denaturation at 95°C for 5 s and the annealing and elongation at 60°C for 34 s. Relative quantification was carried out by normalization of the values relative to those of b-actin. Amplification specificity was examined using the melting curve following the manufacturer’s instructions. Analysis and quantifica- tion using the comparative Ct method were carried out with the ABI Prism 7300 Sequence Detection Software (SDS) version 1.2 (Applied Biosystems).
The data are presented as mean±standard error of the mean (SEM). One-way ANOVA followed by the Bonferroni post-test was performed for statistical analysis of the differences in mRNA expression between reproductive stages. Analyses were performed using GraphPad Prism and SigmaPlot computer software.
Results
Brain and ovarian GnRH mRNA levels
Messenger RNAs for the three GnRH forms were detected in the female pejerrey brains (Fig.1a). Brain pjGnRH mRNA levels were found to be significantly higher in developing females whereas sGnRH was
Table 1 Oligonucleotide primers used for real-time PCR analysis of GnRH and GnRH-R gene expression in pejerrey (Odontesthes bonariensis) females
FwForward,Rvreverse
Primer Nucleotide sequence Length (bp) Tm(°C)
sGnRHFw 50-GGAGGCAAGCAGCAGAGTTATG-30 22 60
sGnRHRv 50-GGCACAGACTGACCTGAACCA-30 21 60
cIIGnRHFw 50-TGAGAGCTGAGAACAAGGTGAGAA-30 24 59
cIIGnRHRv 50-AGCCCCCACGTATAGAAGCA-30 20 58
pjGnRHFw 50-GCGCAGGTTGGAGACGTT-30 18 58
pjGnRHRv 50-CCACAGTGCCCACGTTCTT-30 19 58
pjGnRH-RIFw 50-TCGCAACAAGCCCGTCTTAC-30 20 60
pjGnRH-RIRv 50-GCCATTATCCCCTGCTATTACCT-30 23 58
pjGnRH-RIIFw 50-CATTACCCATCCCGAGGACTT-30 21 59
pjGnRH-RIIRv 50-GTGCCAGTGAGGGACGAAA-30 19 58
bactinFw 50-CTCTGGTCGTACCACTGGTATCG-30 23 60
bactinRv 50-GCAGAGCGTAGCCTTCATAGATG-30 23 60
higher inrestingfemales. The levels of brain cGnRH- II mRNA did not present any significant variation between the three reproductive stages.
Two GnRH variants, namely sGnRH and pjGnRH, were found also to be clearly expressed in ovarian tissue while cGnRH-II mRNA was not detected at all (Fig.1b). Among the two clearly expressed forms, sGnRH had notably higher mRNA expression levels and, in addition, showed a clear expression increase indevelopinggonads compared to those inrestingor spent stages. In contrast, the expression of pjGnRH was low regardless of the reproductive stage.
Brain, pituitary, and ovarian GnRH-R mRNA levels
The mRNAs for the two pjGnRH-R forms were found to be expressed in the female pejerrey brain
(Fig.2a). The form I showed higher mRNA levels in the brain ofrestingfemales when compared to those in the other reproductive stages. The form II mRNA Fig. 1 (a) Brain GnRH mRNA levels in pejerrey (Odontesthes
bonariensis) females with different reproductive status. (b) Ovarian GnRH mRNA levels in pejerrey females with different reproductive status. Values are mean±SEM (n=6). Values are means±SEM (n=6). Different lettersrepresent statis- tically significant (P\0.05) differences between groups for each hormone variant
Fig. 2 (a) Brain pjGnRH-R I and II gene expression in pejerrey females with different reproductive status. (b) Pituitary pjGnRH-R I and pjGnRH-R II gene expression in pejerrey females with different reproductive status. (c) Ovarian pjGnRH-R I and pjGnRH-R II gene expression in pejerrey females with different reproductive status. Values are mean- s±SEM (n=6). Different letters represent statistically significant (P\0.05) differences between groups for each hormone variant
levels showed a modest and not statistically signif- icant increase indevelopingfemale brains.
In contrast to the brain, the mRNA for pjGnRH-R I was not expressed at all in the pituitary gland at any of the reproductive stages (Fig. 2b). On the other hand, pjGnRH-R II mRNA expression levels showed a clear peak in thedevelopingvitellogenic stage and the relative amount of form II mRNA was approx- imately 10-fold higher in the pituitary than in the brain.
The mRNA expression levels of pjGnRH-R I could not be demonstrated in pejerrey ovaries while that of pjGnRH-R II was found in all reproductive stages studied (Fig. 2c). The highest pjGnRH-R II mRNA levels were observed in thedevelopingstage.
Discussion
This study examined the differential mRNA expres- sion levels of three forms of GnRH and two forms of GnRH receptor in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms (sGnRH, pjGnRH, and cGnRH II) while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The mRNA of the different GnRH variants and receptors showed a differential expression according to the reproductive status of the females.
At the brain level, pjGnRH mRNA showed a positive correlation with the GSI%, reinforcing the idea that this form is an important regulator of the reproductive activity in this species (Montaner et al.
2001). This result agrees with the neuroanatomical location of pjGnRH producing neurons since this variant was shown to be mainly expressed in preoptic area neurons (Stefano et al. 2000; Miranda et al.
2003) and was the only form detected in the pituitary gland (Montaner et al. 2001; Somoza et al. 2002b).
Furthermore, Guilgur et al. (2007) have also shown that pjGnRH grouped in the same clade with other hypophysotropic GnRHs (GnRH-I). This is also consistent with physiological studies performed in different bony fish species, i.e., the gilthead sea bream (Sparus aurata; Powell et al. 1994; Gothilf et al. 1996), the European sea bass (Dicentrarchus
labrax; Rodrı´guez et al.2000) and also red sea bream (Pagrus major), black sea bream (Spondyliosoma cantharus), striped knifejaw (Oplegnathus fasciatus) and Nile tilapia (Oreochromis niloticus) (Senthil- kumaran et al. 1999). In all these species, sbGnRH (GnRH-I) was shown to be relevant to the control of gonadotropin synthesis and release. Furthermore, sbGnRH producing neurons, located at the preoptic area and ventral telencephalon, were the main sbGnRH sources to the pituitary; and brain sbGnRH mRNA levels were shown to fluctuate in correlation with ovarian development (Gothilf et al. 1997; Sen- thilkumaran et al. 1999; Rodrı´guez et al. 2000).
The present work also showed elevated sGnRH mRNA levels during early stages of oocyte develop- ment (restingstage) suggesting that this peptide may play a role in early ovarian stages. In pejerrey, neurons producing sGnRH are known to be located almost exclusively in the terminal nerve ganglion (TNG, Stefano et al.2000; Miranda et al.2003) and sGnRH has not been found reaching the pituitary gland of this species (Montaner et al. 2001). Although little is known on the physiological function of the terminal nerve, it is considered to process information from the olfactory bulbs and the retina (Mu¨nz and Claas1987;
Eisthen et al. 2000). In this context, it has been demonstrated that the photoperiod affected sGnRH levels in the barfin flounder (Verasper moseri; Amano et al. 2004), even though this form of GnRH is not involved in gonadotropin secretion in that species.
Moreover, in the Indian major carp (Cirrhinus mrigala), it was suggested that GnRH in the olfactory system may play a major role in the translation of the environmental cues, sending of downstream signals leading to the stimulation of the brain–pituitary–ovary axis (Biju et al.2003). Knowing that photoperiod and water temperature are the two major factors that regulate gonadal maturation in freshwater fish (Bro- mage et al.2001), the high sGnRH mRNA expression at early stages of ovarian development suggests that sGnRH could play a function in the transduction of environmental signals that manifest specifically at the beginning of the reproductive period in pejerrey. As the role of sGnRH in multiple spawners is not well established it would be interesting to study sGnRH mRNA variations between each spawning event during the reproductive season.
In the present study, brain cGnRH-II mRNA levels in pejerrey did not change with the reproductive
status in females. In this context, this peptide seems to be not directly related to the ovarian development in pejerrey. However, cGnRH-II mRNA levels showed correlation with reproduction in pejerrey males (Miranda et al.2007). This fact, together with the wide distribution of cGnRH-II fibers in the brain but not in the pituitary gland (data not shown), suggest the involvement of cGnRH-II in the neuro- logical process related to reproduction as surmised for other teleosts (Oka 1997; Okuzawa et al. 2003).
Taking into account that this peptide was undetect- able in the pituitary of different teleosts (Senthilkumaran et al. 1999; Amano et al. 2002;
Gonza´lez-Martı´nez et al. 2002; Zmora et al. 2002), and cGnRH-II levels in fish brain remain constant during the sexual cycle in some species (Collins et al.
2001; Dufour et al. 1993; Okuzawa et al. 1990;
Senthilkumaran et al. 1999), cGnRH-II does not seem to be directly involved in gonadotropin secre- tion. However, there are also reports suggesting that cGnRH-II may be involved in the regulation of pituitary function in other teleost species: (1) this peptide was identified as the second or third GnRH in the pituitary gland of: the goldfish (Carassius aura- tus; Yu et al.1988), European eel (Anguilla anguilla;
Dufour et al. 1993), tilapia (Weber et al. 1997), striped bass (Holland et al. 2001), and herring (Clupea harengus pallasi; Carolsfeld et al. 2000);
(2) it varied in correlation with luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and gonadal parameters in stripped bass (Holland et al. 2001); and (3) it has also a well-documented gonadotropin-releasing activity (Mylonas and Zohar 2001). Some evidence has been obtained in mammals suggesting that although cGnRH-II does not appear to be a regulator of pituitary gonadotropins, it has a clear influence on reproductive behavior. Adminis- tration of cGnRH-II heightened sexual behavior and inhibited short-term food intake in underfed female shrews,Suncus murinus, and this effect was mediated by cGnRHR-II independently of the hipophysotropic form (Temple et al. 2003; Kauffman and Rissman 2004; Kauffman et al. 2005). Therefore, the physio- logical role(s) of cGnRH-II may be different among species, and more detailed work on the role of this peptide is mandatory for pejerrey in particular and for other species in general.
It has also been reported that the majority of teleost fish species possess at least two types of
GnRH-Rs with distinct brain localizations (Okubo et al.2003; Peter et al.2003; Soga et al.2005; Chen and Fernald 2006). The two GnRH receptors described in pejerrey were shown to be expressed in the brain at all the ovarian stages analyzed.
However, only pjGnRH-R I showed statistically significant increase in mRNA levels in the resting stage when compared to the other gonadal stages. As discussed above, in pejerrey sGnRH and cGnRH-II probably function as neurotransmitters or neuromod- ulators in the brain because their immunoreactive fibers are widely distributed in the brain but not in the pituitary (Stefano et al. 2000; Miranda et al. 2003).
Thus, the correlation between sGnRH mRNA levels and those of pjGnRH-R I suggests that this variant may play a role during the early stages of oocyte maturation being their effects mediated by GnRH-R I in pejerrey brain.
In this context, it is important to note that other piscine GnRH-Rs with high amino acid sequence similarity with that of pjGnRH-R I [e.g., rtGnRH-R from rainbow trout (Oncorhynchus mykiss), 71.4%
identity; GnRH-RGfA from goldfishCarassius aura- tus (72.2%); and GnRH-R2 from Astatotilapia burtoni (80.5%)] were shown to be widespread throughout the brain (Madigou et al. 2000; Peter et al. 2003; Chen and Fernald 2006). Likewise, one of the GnRH receptors characterized in the African catfish (Clarias gariepinus; cfGnRH-R2; 72.3%
sequence identity with pjGnRH-R I) was surmised to have important functions in the brain and be stimulated predominantly by cGnRH-II (Bogerd et al.
2002). In addition, Hajdu´ et al. (2007) clearly dem- onstrated that TNG-GnRH neurons expressed two GnRH-Rs in the dwarf gourami (Colisa lalia). This finding is in good agreement with the presence of sGnRH mRNA in these cells and the electrophysio- logical demonstration of autocrine/paracrine regulation of TNG-GnRH neurones by sGnRH (Abe and Oka2000;2002).
Only pjGnRH-R II was expressed in the pituitary gland of pejerrey. Moreover, the expression of this receptor in female pituitaries was particularly high in the developingstage, supporting the assumption that it could be the GnRH receptor responsible for direct pjGnRH stimulatory actions on synthesis and release of GtHs. Our results agree with reports on other fish species that showed GnRH-Rs variation over the gonadal maturation process (Habibi and Peter 1991;
Alok et al. 2000; Gonza´lez-Martı´nez et al. 2004).
The low expression levels of this receptor type in the brain and the absence of pjGnRH-R I in the pituitary gland supports the notion that they are different receptors not only from a structural point of view but also on a functional basis.
It is now a fact that GnRH mRNA is produced in the gonads of several fish species as: midshipman (Grober et al. 1995); goldfish (Lin and Peter1996);
African cichlid (White and Fernald 1998); sockeye salmon (Oncorhynchus nerka) (Von Schalburg et al.
1999); rainbow trout (Von Schalburg and Sherwood 1999a); seabream (Nabissi et al. 2000); African catfish (Bogerd et al.2002); sea bass (Moncaut et al.
2005), spotted green pufferfish (Ikemoto and Park 2005) and pejerrey (Guilgur et al. 2007). Moreover, GnRH-like peptides have been purified from fish gonads (Habibi et al. 1994; Nabissi et al.1997; Pati and Habibi 1998; Von Schalburg et al. 1999) indi- cating that GnRH mRNA expression is actually translated into functional peptides. Furthermore, there are also indications that GnRH is involved in paracrine / autocrine regulation of gonadal processes such as steroidogenesis (Pati and Habibi 1998) and meiosis (Habibi et al. 1988; Nabissi et al. 1997;
2000; Pati and Habibi2000) in teleost fishes.
The present study also demonstrated the expres- sion of sGnRH, pjGnRH and pjGnRH-R II mRNA in pejerrey ovaries. sGnRH had by far the highest expression levels among the two gonadal GnRH forms and showed marked changes according to the reproductive status. This result agrees with reports that gonadal sGnRH varied during the sexual cycle in rainbow trout (von Schalburg et al.1999; Uzbekova et al. 2001). Interestingly, the highest pjGnRH-R II mRNA levels temporally coincided with the peak expression of sGnRH in the developing stage, suggesting that the expression of this receptor is regulated by sGnRH. In contrast to sGnRH, pjGnRH mRNA was only faintly detected in pejerrey ovaries and did no correlate with the reproductive status of the females. This is a probable indication that this ovarian GnRH form is not related to the gonadal development process in pejerrey.
In conclusion, we demonstrated a correlation between the status of ovarian development and the mRNA expression of GnRH and GnRH-R variants in the brain, pituitary, and ovary of pejerrey. Thus, pjGnRH and pjGnRH-R II could be the key factors in
relation to GtHs secretion and ovarian maturation.
Furthermore, sGnRH and pjGnRH-R I appear to be important during the early stages of ovarian devel- opment, probably in the translation of the environmental signals that cue cyclical reproductive activity. Finally, the present results also strongly support the hypothesis that sGnRH is involved in the regulation of fish ovarian function.
Acknowledgments The authors would like to thank Juan Ignacio Fernandino, Pablo Strobl-Mazzulla, Ricardo Hattori, and Gabriela Carina Lo´pez for their technical assistance This work was supported by grants given to G.M.S (CONICET, PIP
#5425 and ANPCyT PICTR-528) and to C.A.S. (Ministry of Education, Culture, Sports, Science and Technology of Japan
#15201003).
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