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. 2016 Dec 21;11(12):e0168874.
doi: 10.1371/journal.pone.0168874. eCollection 2016.

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development

Feibiao Song  1 , Lanmei Wang  2 , Wenbin Zhu  2 , Jianjun Fu  2 , Juanjuan Dong  1 , Zaijie Dong  1   2
Affiliations

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development

Feibiao Song et al. PLoS One. .

Abstract

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17β-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Nucleotide sequences and the deduced aa sequences of C. carpio Igf3a (A) and Igf3b (B).
The start codons (ATG) and stop codons (TGA) are boxed; the putative signal peptides are underlined (A, B); underlined and bold sequences indicate the polyadenylation sites (AATAAA) (B).
Fig 2
Fig 2. Comparison of Igf3 aa sequences from different species.
Dark shaded areas indicate examples of species with the same aa sites; light shaded areas indicate that more than half of the listed species have the same aa sites. The mature region (B, C, A, D domains) and E domain are underlined with different colors.
Fig 3
Fig 3. Phylogenetic analysis of Igf1, Igf2 and Igf3 in vertebrates.
NJ phylogenetic tree of Igf3 using the deduced aa sequences of C. carpio and the Igf1, Igf2 and Igf3 deduced aa sequences of other species in the GenBank database: Homo sapiens (Igf1a: AAA52538.1, Igf1b: AAA52537.1, Igf2: AAA60088.1), Mus musculus (Igf1: AAH12409.1, Igf2: AAH58615.1), Rattus norvegicus (Igf1: P08025.3, Igf2: XP_008758297.1), D. rerio (Igf1: AAI14263.1, Igf2a: NP_571508.1, Igf2b: NP_001001815.1, Igf3a: ADO16598.1, Igf3b: ADO16599.1), C. carpio (Igf1: AKK31842.1, Igf2a: ADO16599.1, Igf2b: ADQ44896.1, Igf3: obtained in the present study), Carassius auratus (Igf1: AHZ62772.1, Igf2: ACJ37293.1), X. laevis (Igf1: NP_001156865.1, Igf2a: NP_001082128.1, Igf2b: NP_001085129.2, Igf3: NP_001082137.1), X. tropicalis (Igf1: XP_002936875.1, Igf2: AAI56000.1, Igf3: NP_001120418.1), O. niloticus (Igf1: NP_001266432.1, Igf2: NP_001120418.1, Igf3: NP_001266565.1), Ctenopharyngodon idella (Igf1: AGW17294.1, Igf2: NP_001266565.1), Ictalurus punctatus (Igf1: AAZ28918.1, Igf2: NP_001187128.1), Oryzias latipes (Igf1a: XP_011489575.1, Igf1b: XP_004083254.2, Igf2: XP_011491450.1), Dicentrarchus labrax (Igf3: AGB51126.1), K. marmoratus (Igf3: AGA82753.1), and E. coioides (AML84199.1). Numbers at each branch indicate the bootstrap value (%). Bar at the bottom indicates 5% aa divergence in the sequences.
Fig 4
Fig 4. Profile of igf3a (A) and igf3b (B) mRNA expression in adult C. carpio.
Blood (Bl), ovary (Ov), testis (Te), kidney (Ki), intestine (In), liver (Li), skin (Sk), spleen (Sp), heart (He), muscle (Mu), brain (Br).
Fig 5
Fig 5. Expression of C. carpio igf3a (A, B) and igf3b (C, D) mRNA in gonadal development.
Fig 6
Fig 6. Western blot analyses of Igf3 from C. carpio testis and ovary.
Fig 7
Fig 7. Immunohistochemistry localization of Igf3 in C. carpio testis and ovary.
Positive signals of anti-Igf3 immunolabeling are shown in brown. Igf3 was expressed in the spermatogonium (C) and spermatids (E)of the testis. Igf3 was expressed in the follicle cells (D) and granulosa cells of the ovary (F). A negative control for the testis (A) and ovary (B). Sg, spermatogonium; St, spermatids; F, follicle cell; G, granulosa cell.
Fig 8
Fig 8. Effect of E2 on igf3 mRNA expression levels in the testis and ovary.
Y-axis shows data presented as the mean ratios ± SEM between treatment and control groups.
See this image and copyright information in PMC

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