Variations in brain GnRHs and pituitary gonadotropins expression in European eel males in relation with artificial gonad maturation.

D.S. Pearanda1, L. Prez1, X. Espert1, V. Gallego1, S. Baloche2, M. Jover1, S. Dufour2, J.F. Asturiano1
(1)Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnologa Animal. Universidad Politcnica de Valencia. Valencia, Spain. (2)Biologie des Organismes Marins et Ecosystmes, Dpartement des Milieux et Peuplement Aquatiques, Musum National dHiistoire Naturelle. Paris, France.

Introduction

One weekly injection of human chorionic gonadotropin (HCG, 1.5 IU/g fish, Prez et al., 2000) is the common method to obtain gonad maturation in European eel males. The present study evaluated the expression of GnRHs (mGnRH and cGnRH) in several parts of the brain and the expression of gonadotropins in the pituitary during the treatment. Moreover, 11-ketotestosterone (11-KT) plasma levels, testis histology, sperm quality and morphology parameters (eye index, GSI) were analyzed.

Material and Methods

Three tanks were used to maintain 150 males of European eel, which were weekly treated with HCG. Ten fish were sacrificed every week to obtain brains, pituitaries, blood and sperm samples. Other parameters were noted, as gonadosomatic index (GSI), eye index or sperm motility (Fig. 1).

Figure 1. UPV aquaculture facilities. Intraperitoneal HCG administration, sperm extraction and and evaluation of sperm samples.

The expression of pituitary gonadotropins was analyzed by rq RT-PCR (Aroua et al., 2007) , the plasma level of 11-KT by ELISA , and mGnRH and cGnRH-II expression were analyzed by rq RT-PCR in several parts of the brain (Fig. 2, Montero et al. 1994), but just from 3th week on.

Pytuitary mGnRH cGnRH

Figure 2. Blood extraction for 11-KT analysis by ELISA, and the process to obtain the eel brain (divided in different parts). The expression of gonadotropins and GnRHs was performed by qr RT-PCR using a Light Cycler System.

Results
GnRHs variations were found in diencephalon and mesencephalon. cGnRH showed a significant decrease in 4th week, followed by no significant variations. The highest value of mGnRH was found in the 3th week, and was followed by a decrease and a significant increase of the expression during last weeks of treatment (Fig. 3). The FSH- expression showed a dramatic decrease with only one hormone injection, and after 4 weeks was close to zero. The -LH subunit expression showed a progressive increase during the first 7 weeks, showing a positive correlation with eye index and GSI (Fig. 4). Testis histology evidenced important changes during the beginning of the treatment (Fig. 6). Just spermatogonia were observed at 2nd week, but all the stages of development (except spermatoza) were found at 3rd, coinciding with an important increase of 11-KT (Fig.5). Spermatozoa were detected in the testis at 4th week, as well as an important GSI increase (Fig.5).

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Figure 3. Relative expression of GnRHs in different parts of the eel brain. Letters show Figure 4. Relative expression of gonadotropins in pytuitary and evolution of Figure 5.Level of 11-KT in plasma and significant difference. morphology parameters during the treatment. Letters and asterisks show GSI during treatment. Letters and significant difference. asterisks show significant differences.

Figure 6. Testis histology in the 2nd ,3th and 4th weeks of the experiment (a: spermatogonium. b: spermatocyte I. c: spermatocyte II. d: spermatid. e: spermatozoa).

Conclusion

LH seems the gonadotropin responsible of gonad maturation, showing a positive correlation with reproductive parameters (GSI or EI). Significant differences were found in mesencephalon-diencephalon for GnRHs expression. The 11KT can play an important role during spermatogenesis. After a single dose of HCG, 11KT plasma level increased 4 times, but showed a more important increase at 3th week of treatment coinciding with the appearance of all cellular types (except spermatozoa) in the gonad. Spermatozoa were observed at 4th week, as well as an important GSI increase, due to possible testis hydration.
Acknowledgements Supported by the Generalitat Valenciana (GV04A508, ACOMP06/193, and a grant to D.S. Pearanda). The eel farm Valenciana de Acuicuktura, S.A. (Puzol, Valencia) provided the male eels used in these experiments.
References - Prez et al., 2000. Induction of maturation and spermiation in the male European eel (Anguilla anguilla). Assessment of sperm quality throughout treatment. Journal of Fish Biology, 57: 1488-1504 - Montero M., Vidal B., King J.A., Tramu G., Vandesande F., Dufour S., Kah O., 1994. Immunocytochemical localization of mammalian GnRH (gonadotropin-realising hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.). Journal of Chemical Neuroanatomy 7, 227-241. - Aroua S., Weltzien F-A., Le Belle N., Dufour S., 2007. Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids. General and Comparative Endocrinology 153, 333-343.