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Enhanced expression of β-thymosin mRNA in the ovary of GnRH analog or estradiol-17β-treated paradise fish, Macropodus opercularis
Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
Enhanced expression of b-thymosin mRNA in the ovary of GnRH analog or estradiol-17b-treated paradise fish, Macropodus opercularis V. Anathy, S. Kirankumar, T.J. Pandian* Department of Genetics, Center for Advanced Studies in Functional Genomics, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India Received 23 May 2002; received in revised form 10 September 2002; accepted 2 December 2002
Abstract cDNA clones were isolated as expressed sequence tags (ESTs) from the ovarian cDNA library of Macropodus opercularis. The EST sequences showed similarity with many housekeeping genes and ribosomal proteins. One of the ESTs showed similarity to b-thymosin, a 5-kDa polypeptide expressed under different physiological conditions. The cDNA corresponding to b-thymosin of M. opercularis is 368 bp in length and codes for a putative polypeptide of 42 amino acids. Multiple alignment of the deduced amino acid sequence showed 61% similarity with piscine b-thymosins and 56% similarity with mammalian b-thymosins. Administration of a gonadotropin releasing hormone analog or estradiol-17b induced an increase in the gonadosomatic index, oocyte diameter and also enhanced expression of bthymosin m-RNA in the recrudizing ovary. This report indicates that both GnRH analog and E2 might induce similar pathways for the differentiation of ovarian cells for the maturation of oocytes. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: b-Thymosin mRNA; Macropodus opercularis; Sequence and phylogeny; Estradiol-17b; GnRH analog; Steroidogenic pathway; Oocyte differentiation; Gonadosomatic index
1. Introduction Reproduction is a highly integrative function, whose success depends on a delicate balance between the hormonal status of the individual and surrounding environment. Sex steroids have potent effect on the process of sex differentiation and gametogenesis in all vertebrates (Nagahama, 2000). As their profound, multiple effects have been described, teleostean fishes provide excellent models for investigating the basic hormonal mechanism. The Chinese paradise fish (Macropodus *Corresponding author. Tel.: q91-452-2458212; fax: q91452-2459139. E-mail address: [email protected] (T.J. Pandian).
opercularis) exhibits a peculiar phenomenon, in which the sex ratio is tilted in favour of females with increasing rearing density (Francis, 1983). bthymosins are a family of highly conserved polar 5-kDa polypeptides present in high concentrations in almost every cell. They play an important role in the organization of the cytoskeleton sequestering G-actin. Several biological effects are attributed to thymosins, viz.: induction of chemotaxis, angiogenesis and inhibition of inflammation and bone marrow stem cell proliferation (Huff et al., 2001). The thymosins are classified into three main groups according to their isoelectric points: athymosins below pH 5.0, b-thymosins between pH 5.0 and 7.0, and g-thymosins above pH 7.0.
1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(02)00367-7
640
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
Thymosin b4 is found to effect the hypothalamus and pituitary and to induce the release of GnRH (Rebar et al., 1981). Studies on the changes in mRNA expression of b-thymosins in cells and tissues have been a topic of interest to several groups. Friedman et al. (1984) reported interferon induced increase of thymosin b4 mRNA in human neuroblastoma and lymphoblastoid cell lines. Gondo et al. (1987) suggested a role for thymosin b4 in host defense. Likewise, the plasma concentrations of thymosin b4 are reported to be elevated during molting in domestic hens (Dickerman et al., 1992) and during the estrual period in heifers (Wise and Maurer, 1991). These studies show that mRNA as well as protein levels of b-thymosins can be altered rapidly by several different stimuli and also during differentiation (Hall et al., 1991; Huff et al., 2001). Gonadotropin and estradiol-17b (E2) regulate reproduction either directly or through related signaling pathways by programming or imprinting genes involved in cell proliferation, differentiation or survival. E2 is widely used for sex reversal studies in teleosts (Piferrer, 2001). Also the presence of E2-mimics in the industrial pollutants are known to alter the reproductive cycle in the wild population of fish (Devlin and Nagahama, 2002). Hence, a thorough knowledge on gonadotropin or E2 regulated genes in vertebrates will give us valuable information on the mechanism of action of these physiologically important hormones. Keeping this in mind, a study on the in vivo expression of b-thymosin was initiated in the recrudizing ovary in response to exogenously administered GnRH analog or E2. This communication is perhaps the first to show that the b-thymosin may be induced in the recrudizing ovary of a teleost and thereby indicates a role for b-thymosin in the reproductive pathway of the fish. 2. Material and methods 2.1. Animals Matured Chinese paradise fish were collected from the local ornamental fish dealer and were maintained in the well aerated freshwater aquaria under 14L: 10D photoperiodism. They were fed with tubifex and dry pelleted feed. Gonadally recrudizing individuals were selected for the experimental treatment and anesthetized in clove oil (50 mlyl of water) before killing by cervical dislocation.
2.2. Construction of cDNA library Total cellular RNA was extracted from the recrudizing ovary of M. opercularis using TRIZOL reagent following the instructions provided in the kit. The RNA was analyzed by denaturing formaldehyde (agarose) gel electrophoresis and quantified spectrophotometrically. Poly Aq RNA was purified from total cellular RNA by affinity chromatography with oligo dT cellulose, following the standard protocol (Sambrook et al., 1989). Using this poly Aq RNA as template, a cDNA library was constructed with l ZAP cDNA library cloning kit, as per the manufacturer’s protocol (Stratagene). The recombinant phages were grown in E. coli XLI blue MRF9 cells and excised randomly to generate large number of cDNA clones for the analysis of expressed sequence tags (ESTs) from the ovary of M. opercularis. 2.3. DNA sequencing and sequence analysis The nucleotide sequences of the ESTs were determined by Sanger’s dideoxy chain termination method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All the computational analysis of the sequence was made using GCG-9.1, Wisconsin package. 2.4. GnRH analog or E2 induction and Northern blotting Ten recrudizing females reared in equal numbers in two different aquaria were individually induced by a prime dose of GnRH analog Ovaprim (Syndel laboratories Ltd, Vancouver, Canada) (2 Uyg body wt.) or E2 (Sigma, St. Louis, MO) (1 mgyg body wt.). Stock solution of E2 was prepared in absolute alcohol (carrier) at a concentration of 1 mgyml. Five females reared in a separate aquaria were individually injected with carrier and these animals served as control. The animals were anesthetized and individually weighed to the nearest 0.1 mg. Ovaries were dissected out both from the treated and the control animals 6 h. after the injection and weighed to the nearest 0.1 mg. From the ovary few eggs were separated out and the egg diameter was measured using the micrometer. Gonadosomatic index (GSI) was calculated by expressing gonad mass as a percentage of body mass. The ovarian tissue was homogenised in TRIZOL reagent and total RNA was isolated according to the instructions in the kit. Transcripts were sepa-
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
641
Fig. 1. cDNA and putative amino acid sequence of the b-thymosin of the M. opercularis. (GenBank Acc. No. AF452101) The putative actin-binding region of the polypeptide is shown in bold letters.
rated in a formaldehyde–agarose gels. Four microgram of total RNA was loaded per lane. Northern blot hybridization was performed following the protocols of Sambrook et al. (1989) and the digoxigenin (DIG) system user guide for filter hybridization (Boehringer Mannheim, Mannheim, Germany). RNA was transferred to a nylon membrane and UV crosslinked. b-Thymosin cDNA (370 bp) was labelled with DIG and hybridization was performed at 50 8C overnight in a Techne hybridization tube, followed by several washes at a maximal stringency of 0.1=SSC, 0.1% SDS at 50 8C. Hybridized probes were visualised using anti-DIG-AP Fab fragments (diluted 1:10 000 in Boehringer buffer) and the chemoluminescence reagent CDP-Star. 2.5. Statistical analysis One way ANOVA was used to compare the GSI and oocyte diameter among the control, GnRH analog-treated and E2-treated fish using the F-test with 95% CL (Zar, 1984). The differences were regarded significant when P-0.001.
3. Results and discussion 3.1. Cloning and sequencing of b-thymosin cDNA In a search for genes that are transiently expressed in the recrudizing ovary of the Chinese paradise fish, the cDNA library clones were randomly excised and ESTs were generated. One of them showed sequence similarity with b-thymosin of other organisms. The b-thymosin cDNA of M. opercularis is 370 bp in length, has a 59 UTR of 56 bps, a 126 bps ORF and a 39 UTR of 150 bps (Fig. 1). The ORF codes for a putative 42-aminoacid polypeptide that has all the typical characteristics of b-thymosin like a conserved helix-forming region and an actin-binding domain followed by another helix-forming region (Fig. 2). These regions are conserved throughout the animal kingdom. 3.2. Sequence analysis and phylogeny Comparison of deduced amino-acid sequences of four mammalian and seven piscine b-thymosins showed the conserved helix–actin binding–helix
642
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
Fig. 2. Multiple alignment of the deduced amino acid sequences of b-thymosins. The conserved residues are marked with the sign of asterisk (*). The residues of the putative helix-forming regions and the actin-binding domain have been marked with bold and italic letters, respectively. Box represents the conserved b-thymosin signature (actin-binding domain).
Fig. 3. Phylogram showing the evolutionary relationship based on b-thymosin peptide sequences.
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
643
regions. The LKKTETQEK a signature sequence showed high degree of conservation in all these organisms (Fig. 2). Multiple alignment of amino acid sequences indicated that the b-thymosin of the Chinese paradise fish shares 59–62% similarity with other piscine sequences (Japanese seaperch, common carp, zebrafish, rainbow trout) and 55– 56% similarity with mammalian sequences (rat, mouse, human, bull). When the amino-acid sequence of b-thymosin was compared with the corresponding common carp a and b sequences the similarity was 49 and 61%, respectively, indicating the existence of two different forms of piscine b-thymosin. A phylogram was deduced by the neighbour-joining method (Fig. 3). The phylogram was derived from the putative peptide sequences of available b-thymosins. The phylogenetic tree showed the separate clustering of the piscine and mammalian sequences at equal distance. 3.3. mRNA expression in the recrudescent ovary of GnRH analog or E2 treated fish The GSI values of both GnRH analog- or E2treated fish were significantly (P-0.001) higher than that of the control. However there was no significant difference in the GSI among the treated groups. Likewise, there was significant increase (50–80 mm) in the diameter of oocytes in treated individuals compared to control. But there was a significant difference in the oocyte diameter of the GnRH analog- and E2-treated fish. Also in the treated individuals the oocytes became translucent in color (Table 1). The Northern blot analysis showed that the administration of either GnRH analog or E2 in M. opercularis led to a ;2–3-fold increase in the expression of b-thymosin in the recrudizing ovary (Fig. 4). Incidentally, this observation confirms the earlier report showing an increase of thymosin b10 mRNA levels in immature ovaries of rat treated
Fig. 4. Northern blot analysis of b-thymosin mRNA expressed in tha ovary of M. opercularis. Lane 1, control; lane 2, treated with GnRH analog; lane 3, treated with E2.
with pregnant mare’s serum gonadotropin (Hall et al., 1991). However, that the administration of E2 induced an increase in the b-thymosin mRNA in the recrudizing ovary is an observation, which has not so far been reported (Fig. 4). Estrogens are found to modulate the circulating levels of thymosin b4 in women (Suh et al., 1985). Ford et al. (1990) showed elevated levels of thymosin b4 in the serum during follicular development in the estrous cycle of pigs. Evidently, there are similar responses in b-thymosin mRNA expression to these two different hormonal stimuli. Fluctuating levels of GnRHyE2 over the reproductive cycle represent a very efficient way to synchronize hormonal responses at all levels of the organism, including at the brain and pituitary levels (Kah et al., 2000). During development, it is believed that
Table 1 Characteristics of the recrudizing ovary induced with GnRH analog or E2 Treatment Control GnRH analog-treated E2-treated
GSI a
8.16"1.22 11.22"1.04b 10.70"0.75b
Oocyte color
Oocyte diameter (mm)
Brown Translucent Translucent, light yellow
632"20c 711"10d 680"20d
Each value is the mean"S.D. of five samples. Values followed by different letters differ significantly (P-0.001) (ANOVA).
644
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
and E2 induction of (genes) of the fish (ovary) will lead to the differentiation and dedifferentiation of the ovarian cells either for maturation of gametes or for sex reversal (Nagahama, 2000) (Fig. 5). Our study indicates the enhanced expression of b-thymosin mRNA in the ovary in response to either GnRH analog or E2 treatment indicating that these two hormones are interlinked in two different pathways; they may independently induce b-thymosin expression which may lead to maturation of gametesysex reversal. Further investigation on the downstream molecules is in progress to throw light on the role of GnRH, E2 and b-thymosin and their interrelationships on maturation of gametesysex reversal. Acknowledgments
Fig. 5. Expression of b-thymosin (j) seems to be involved in the differentiation and dedifferentiation, and may be acting as one of the key factors in the process of sex reversalymaturation of oocyte in M. opercularis.
The authors thank an unknown referee for a number of useful suggestions. They also thank Council of Scientific and Industrial Research (CSIR), New Delhi for the financial support. For providing sequencing and computational facilities, the CAS in Functional Genomics and the Bioinformatics centre of our university are gratefully acknowledged. References
GnRHyE2 participate in the organization and maturation of the neuro-endocrine systems and synchronize them during sexual maturation. This synchronization may be brought out by b-thymosin like small polypeptides, which may finally lead to ovarian cell differentiation and proliferation, as in the case of T-lymphocyte differentiation, wherein b-thymosin plays the mediatory role (Low et al., 1981; Wise et al., 1991). During the physiological transitional state wherein the oocyte enters into the maturation phase from the growth phase, a shift in the steroidogenic pathway occurs, leading to highlevel production of 17a,20b-DP instead of E2. This shift, a prerequisite for the growing oocyte to enter the maturation phase, requires a complex, integrated network of gene regulation involving cell specificity, hormonal regulation and developmental patterning (Nagahama, 2000). The central role of GnRH, a neuropeptide, is to mediate the release of gonadotropins from the pituitary, but is also expressed in the gonads during the periods of maturation, when they may play an autocriney paracrine role (Kah et al., 2000). GnRH analog
Devlin, R.H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191–364. Dickerman, R.W., Wise, T.H., Bahr, J.M., 1992. Effect of ovarian regression and molt on plasma concentrations of thymosin beta 4 in domestic hens (Gallus domesticus). Domest. Anim. Endocrinol. 9, 297–304. Ford, J.J., Vakharia, D.D., Anderson, L.L., Klindt, J., 1990. Thymosin-beta 4 concentrations during the estrous cycle and after hypophyseal stalk transection of female pigs. Proc. Soc. Exp. Biol. Med. 193, 185–189. Francis, C.R., 1983. The effects of bidirectional selection for social dominance on agonistic behaviour and sex ratios in the paradise fish (Macropodus opercularis). Behaviour 90, 25–45. Friedman, R.L., Manly, S.P., McMahon, M., Kerr, I.M., Stark, G.R., 1984. Transcriptional and post-transcriptional regulation of interferon-induced gene expression in human cells. Cell 38, 745–755. Gondo, H., Kudo, J., White, J.W., Barr, C., Selvanayagam, P., Saunders, G.F., 1987. Differential expression of the human thymosin b4 gene in lymphocytes, macrophages and granulocytes. J. Immunol. 139, 3840–3848. Hall, A.K., Aten, R., Behrman, H.R., 1991. Thymosin gene expression is modulated by pregnant mare’s serum gonadotropin, human chorionic gonadotropin, anf prostaglandin
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645 F2 a in the immature rat ovary. Endocrinology 128, 951–957. Huff, T., Muller, C.S.G., Otto, A.M., Netzker, R., Hannappel, E., 2001. Thymosins, small acidic peptides with multiple functions. Int. J. Biochem. Cell Biol. 33, 205–220. Kah, O., Madigou, T., Mazurais, D., Le Drean, G., 2000. Aspects of the central regulation of reproduction in teleost fish. In: Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E., Stefansson, S.O. (Eds.), Proceedings of the Sixth International Symposium in Reproductive Physiology of Fish. University of Bergen, pp. 27–34. Low, T.L.K., Hu, S.K., Goldstein, A.L., 1981. Complete amino acid sequence of bovine thymosin b4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations. Proc. Natl Acad. Sci. USA 78, 1162–1166. Nagahama, Y., 2000. Gonadal steroid hormones: major regulations of gonadal sex differentiation and gametogenesis in fish. In: Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E., Stefansson, S.O. (Eds.), Proceedings of the Sixth International Symposium on Reproductive Physiology of Fish. University of Bergen, pp. 211–222.
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Piferrer, F., 2001. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197, 229–281. Rebar, R.W., Miyake, A.M., Low, T.L.K., Goldstein, A.L., 1981. Thymosin stimulates secretion of luteinizing hormonereleasing factor. Science 214, 669–671. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Suh, B.Y., Naylor, P.H., Goldstein, A.L., Rebar, R.W., 1985. Modulation of thymosin beta 4 by estrogen. Am. J. Obstetr. Gynecol. 151, 544–549. Wise, T., Klindt, J., Macdonald, G.J., Ford, J.J., 1991. Effects of neonatal sexual differentiation, growth hormone and testosterone on thymic weights and thymosin beta 4 in hypophysectomized rats. J. Reprod. Immunol. 19, 43–54. Wise, T., Maurer, R.R., 1991. Characterization of thymosin alpha 1 and beta 4 during the bovine estrual period: effects of elevated estradiol and progestin. Biol. Reprod. 45, 57–63. Zar, H.J., 1984. Biostatistical Analysis. Prentice-Hall International, New Jersey.
Enhanced expression of b-thymosin mRNA in the ovary of GnRH analog or estradiol-17b-treated paradise fish, Macropodus opercularis V. Anathy, S. Kirankumar, T.J. Pandian* Department of Genetics, Center for Advanced Studies in Functional Genomics, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India Received 23 May 2002; received in revised form 10 September 2002; accepted 2 December 2002
Abstract cDNA clones were isolated as expressed sequence tags (ESTs) from the ovarian cDNA library of Macropodus opercularis. The EST sequences showed similarity with many housekeeping genes and ribosomal proteins. One of the ESTs showed similarity to b-thymosin, a 5-kDa polypeptide expressed under different physiological conditions. The cDNA corresponding to b-thymosin of M. opercularis is 368 bp in length and codes for a putative polypeptide of 42 amino acids. Multiple alignment of the deduced amino acid sequence showed 61% similarity with piscine b-thymosins and 56% similarity with mammalian b-thymosins. Administration of a gonadotropin releasing hormone analog or estradiol-17b induced an increase in the gonadosomatic index, oocyte diameter and also enhanced expression of bthymosin m-RNA in the recrudizing ovary. This report indicates that both GnRH analog and E2 might induce similar pathways for the differentiation of ovarian cells for the maturation of oocytes. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: b-Thymosin mRNA; Macropodus opercularis; Sequence and phylogeny; Estradiol-17b; GnRH analog; Steroidogenic pathway; Oocyte differentiation; Gonadosomatic index
1. Introduction Reproduction is a highly integrative function, whose success depends on a delicate balance between the hormonal status of the individual and surrounding environment. Sex steroids have potent effect on the process of sex differentiation and gametogenesis in all vertebrates (Nagahama, 2000). As their profound, multiple effects have been described, teleostean fishes provide excellent models for investigating the basic hormonal mechanism. The Chinese paradise fish (Macropodus *Corresponding author. Tel.: q91-452-2458212; fax: q91452-2459139. E-mail address: [email protected] (T.J. Pandian).
opercularis) exhibits a peculiar phenomenon, in which the sex ratio is tilted in favour of females with increasing rearing density (Francis, 1983). bthymosins are a family of highly conserved polar 5-kDa polypeptides present in high concentrations in almost every cell. They play an important role in the organization of the cytoskeleton sequestering G-actin. Several biological effects are attributed to thymosins, viz.: induction of chemotaxis, angiogenesis and inhibition of inflammation and bone marrow stem cell proliferation (Huff et al., 2001). The thymosins are classified into three main groups according to their isoelectric points: athymosins below pH 5.0, b-thymosins between pH 5.0 and 7.0, and g-thymosins above pH 7.0.
1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(02)00367-7
640
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
Thymosin b4 is found to effect the hypothalamus and pituitary and to induce the release of GnRH (Rebar et al., 1981). Studies on the changes in mRNA expression of b-thymosins in cells and tissues have been a topic of interest to several groups. Friedman et al. (1984) reported interferon induced increase of thymosin b4 mRNA in human neuroblastoma and lymphoblastoid cell lines. Gondo et al. (1987) suggested a role for thymosin b4 in host defense. Likewise, the plasma concentrations of thymosin b4 are reported to be elevated during molting in domestic hens (Dickerman et al., 1992) and during the estrual period in heifers (Wise and Maurer, 1991). These studies show that mRNA as well as protein levels of b-thymosins can be altered rapidly by several different stimuli and also during differentiation (Hall et al., 1991; Huff et al., 2001). Gonadotropin and estradiol-17b (E2) regulate reproduction either directly or through related signaling pathways by programming or imprinting genes involved in cell proliferation, differentiation or survival. E2 is widely used for sex reversal studies in teleosts (Piferrer, 2001). Also the presence of E2-mimics in the industrial pollutants are known to alter the reproductive cycle in the wild population of fish (Devlin and Nagahama, 2002). Hence, a thorough knowledge on gonadotropin or E2 regulated genes in vertebrates will give us valuable information on the mechanism of action of these physiologically important hormones. Keeping this in mind, a study on the in vivo expression of b-thymosin was initiated in the recrudizing ovary in response to exogenously administered GnRH analog or E2. This communication is perhaps the first to show that the b-thymosin may be induced in the recrudizing ovary of a teleost and thereby indicates a role for b-thymosin in the reproductive pathway of the fish. 2. Material and methods 2.1. Animals Matured Chinese paradise fish were collected from the local ornamental fish dealer and were maintained in the well aerated freshwater aquaria under 14L: 10D photoperiodism. They were fed with tubifex and dry pelleted feed. Gonadally recrudizing individuals were selected for the experimental treatment and anesthetized in clove oil (50 mlyl of water) before killing by cervical dislocation.
2.2. Construction of cDNA library Total cellular RNA was extracted from the recrudizing ovary of M. opercularis using TRIZOL reagent following the instructions provided in the kit. The RNA was analyzed by denaturing formaldehyde (agarose) gel electrophoresis and quantified spectrophotometrically. Poly Aq RNA was purified from total cellular RNA by affinity chromatography with oligo dT cellulose, following the standard protocol (Sambrook et al., 1989). Using this poly Aq RNA as template, a cDNA library was constructed with l ZAP cDNA library cloning kit, as per the manufacturer’s protocol (Stratagene). The recombinant phages were grown in E. coli XLI blue MRF9 cells and excised randomly to generate large number of cDNA clones for the analysis of expressed sequence tags (ESTs) from the ovary of M. opercularis. 2.3. DNA sequencing and sequence analysis The nucleotide sequences of the ESTs were determined by Sanger’s dideoxy chain termination method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All the computational analysis of the sequence was made using GCG-9.1, Wisconsin package. 2.4. GnRH analog or E2 induction and Northern blotting Ten recrudizing females reared in equal numbers in two different aquaria were individually induced by a prime dose of GnRH analog Ovaprim (Syndel laboratories Ltd, Vancouver, Canada) (2 Uyg body wt.) or E2 (Sigma, St. Louis, MO) (1 mgyg body wt.). Stock solution of E2 was prepared in absolute alcohol (carrier) at a concentration of 1 mgyml. Five females reared in a separate aquaria were individually injected with carrier and these animals served as control. The animals were anesthetized and individually weighed to the nearest 0.1 mg. Ovaries were dissected out both from the treated and the control animals 6 h. after the injection and weighed to the nearest 0.1 mg. From the ovary few eggs were separated out and the egg diameter was measured using the micrometer. Gonadosomatic index (GSI) was calculated by expressing gonad mass as a percentage of body mass. The ovarian tissue was homogenised in TRIZOL reagent and total RNA was isolated according to the instructions in the kit. Transcripts were sepa-
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
641
Fig. 1. cDNA and putative amino acid sequence of the b-thymosin of the M. opercularis. (GenBank Acc. No. AF452101) The putative actin-binding region of the polypeptide is shown in bold letters.
rated in a formaldehyde–agarose gels. Four microgram of total RNA was loaded per lane. Northern blot hybridization was performed following the protocols of Sambrook et al. (1989) and the digoxigenin (DIG) system user guide for filter hybridization (Boehringer Mannheim, Mannheim, Germany). RNA was transferred to a nylon membrane and UV crosslinked. b-Thymosin cDNA (370 bp) was labelled with DIG and hybridization was performed at 50 8C overnight in a Techne hybridization tube, followed by several washes at a maximal stringency of 0.1=SSC, 0.1% SDS at 50 8C. Hybridized probes were visualised using anti-DIG-AP Fab fragments (diluted 1:10 000 in Boehringer buffer) and the chemoluminescence reagent CDP-Star. 2.5. Statistical analysis One way ANOVA was used to compare the GSI and oocyte diameter among the control, GnRH analog-treated and E2-treated fish using the F-test with 95% CL (Zar, 1984). The differences were regarded significant when P-0.001.
3. Results and discussion 3.1. Cloning and sequencing of b-thymosin cDNA In a search for genes that are transiently expressed in the recrudizing ovary of the Chinese paradise fish, the cDNA library clones were randomly excised and ESTs were generated. One of them showed sequence similarity with b-thymosin of other organisms. The b-thymosin cDNA of M. opercularis is 370 bp in length, has a 59 UTR of 56 bps, a 126 bps ORF and a 39 UTR of 150 bps (Fig. 1). The ORF codes for a putative 42-aminoacid polypeptide that has all the typical characteristics of b-thymosin like a conserved helix-forming region and an actin-binding domain followed by another helix-forming region (Fig. 2). These regions are conserved throughout the animal kingdom. 3.2. Sequence analysis and phylogeny Comparison of deduced amino-acid sequences of four mammalian and seven piscine b-thymosins showed the conserved helix–actin binding–helix
642
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
Fig. 2. Multiple alignment of the deduced amino acid sequences of b-thymosins. The conserved residues are marked with the sign of asterisk (*). The residues of the putative helix-forming regions and the actin-binding domain have been marked with bold and italic letters, respectively. Box represents the conserved b-thymosin signature (actin-binding domain).
Fig. 3. Phylogram showing the evolutionary relationship based on b-thymosin peptide sequences.
V. Anathy et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 639–645
643
regions. The LKKTETQEK a signature sequence showed high degree of conservation in all these organisms (Fig. 2). Multiple alignment of amino acid sequences indicated that the b-thymosin of the Chinese paradise fish shares 59–62% similarity with other piscine sequences (Japanese seaperch, common carp, zebrafish, rainbow trout) and 55– 56% similarity with mammalian sequences (rat, mouse, human, bull). When the amino-acid sequence of b-thymosin was compared with the corresponding common carp a and b sequences the similarity was 49 and 61%, respectively, indicating the existence of two different forms of piscine b-thymosin. A phylogram was deduced by the neighbour-joining method (Fig. 3). The phylogram was derived from the putative peptide sequences of available b-thymosins. The phylogenetic tree showed the separate clustering of the piscine and mammalian sequences at equal distance. 3.3. mRNA expression in the recrudescent ovary of GnRH analog or E2 treated fish The GSI values of both GnRH analog- or E2treated fish were significantly (P-0.001) higher than that of the control. However there was no significant difference in the GSI among the treated groups. Likewise, there was significant increase (50–80 mm) in the diameter of oocytes in treated individuals compared to control. But there was a significant difference in the oocyte diameter of the GnRH analog- and E2-treated fish. Also in the treated individuals the oocytes became translucent in color (Table 1). The Northern blot analysis showed that the administration of either GnRH analog or E2 in M. opercularis led to a ;2–3-fold increase in the expression of b-thymosin in the recrudizing ovary (Fig. 4). Incidentally, this observation confirms the earlier report showing an increase of thymosin b10 mRNA levels in immature ovaries of rat treated
Fig. 4. Northern blot analysis of b-thymosin mRNA expressed in tha ovary of M. opercularis. Lane 1, control; lane 2, treated with GnRH analog; lane 3, treated with E2.
with pregnant mare’s serum gonadotropin (Hall et al., 1991). However, that the administration of E2 induced an increase in the b-thymosin mRNA in the recrudizing ovary is an observation, which has not so far been reported (Fig. 4). Estrogens are found to modulate the circulating levels of thymosin b4 in women (Suh et al., 1985). Ford et al. (1990) showed elevated levels of thymosin b4 in the serum during follicular development in the estrous cycle of pigs. Evidently, there are similar responses in b-thymosin mRNA expression to these two different hormonal stimuli. Fluctuating levels of GnRHyE2 over the reproductive cycle represent a very efficient way to synchronize hormonal responses at all levels of the organism, including at the brain and pituitary levels (Kah et al., 2000). During development, it is believed that
Table 1 Characteristics of the recrudizing ovary induced with GnRH analog or E2 Treatment Control GnRH analog-treated E2-treated
GSI a
8.16"1.22 11.22"1.04b 10.70"0.75b
Oocyte color
Oocyte diameter (mm)
Brown Translucent Translucent, light yellow
632"20c 711"10d 680"20d
Each value is the mean"S.D. of five samples. Values followed by different letters differ significantly (P-0.001) (ANOVA).
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and E2 induction of (genes) of the fish (ovary) will lead to the differentiation and dedifferentiation of the ovarian cells either for maturation of gametes or for sex reversal (Nagahama, 2000) (Fig. 5). Our study indicates the enhanced expression of b-thymosin mRNA in the ovary in response to either GnRH analog or E2 treatment indicating that these two hormones are interlinked in two different pathways; they may independently induce b-thymosin expression which may lead to maturation of gametesysex reversal. Further investigation on the downstream molecules is in progress to throw light on the role of GnRH, E2 and b-thymosin and their interrelationships on maturation of gametesysex reversal. Acknowledgments
Fig. 5. Expression of b-thymosin (j) seems to be involved in the differentiation and dedifferentiation, and may be acting as one of the key factors in the process of sex reversalymaturation of oocyte in M. opercularis.
The authors thank an unknown referee for a number of useful suggestions. They also thank Council of Scientific and Industrial Research (CSIR), New Delhi for the financial support. For providing sequencing and computational facilities, the CAS in Functional Genomics and the Bioinformatics centre of our university are gratefully acknowledged. References
GnRHyE2 participate in the organization and maturation of the neuro-endocrine systems and synchronize them during sexual maturation. This synchronization may be brought out by b-thymosin like small polypeptides, which may finally lead to ovarian cell differentiation and proliferation, as in the case of T-lymphocyte differentiation, wherein b-thymosin plays the mediatory role (Low et al., 1981; Wise et al., 1991). During the physiological transitional state wherein the oocyte enters into the maturation phase from the growth phase, a shift in the steroidogenic pathway occurs, leading to highlevel production of 17a,20b-DP instead of E2. This shift, a prerequisite for the growing oocyte to enter the maturation phase, requires a complex, integrated network of gene regulation involving cell specificity, hormonal regulation and developmental patterning (Nagahama, 2000). The central role of GnRH, a neuropeptide, is to mediate the release of gonadotropins from the pituitary, but is also expressed in the gonads during the periods of maturation, when they may play an autocriney paracrine role (Kah et al., 2000). GnRH analog
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