Differential Expression of Duplicate Insulin-like Growth Factor-1 Receptors (igf1rs) in Medaka Gonads

Abstract

Insulin-like growth factor-1 receptors (igf1rs) play important roles in regulating development, differentiation, and proliferation in diverse organisms. In the present study, subtypes of medaka igf1r, igf1ra, and igf1rb were isolated and characterized. RT-PCR results showed that igf1ra and igf1rb mRNA were expressed in all tissues and throughout embryogenesis. Using real-time PCR, the differential expression of igf1ra and igf1rb mRNA during folliculogenesis was observed. The results of in situ hybridization (ISH) revealed that both of them were expressed in ovarian follicles at different stages, and igf1rb was also expressed in theca cells and granulosa cells. In the testis, both igf1ra and igf1rb mRNA were highly expressed in sperm, while igf1rb mRNA was also obviously detected in spermatogonia. In addition, igf1ra mRNA was also present in Leydig cells in contrast to the distribution of igf1rb mRNA in Sertoli cells. Collectively, we demonstrated that differential igf1rs RNA expression identifies medaka meiotic germ cells and somatic cells of both sexes. These findings highlight the importance of the igf system in the development of fish gonads.

Keywords: gene expression; igf1rs; medaka; ovary; testis.

Figure 1

Multiple sequence alignment of medaka Igf1rs. The alignment sequences show the intracellular protein tyrosine kinase domain. Conserved regions between species are highlighted. The length and percentage identity values of Igf1rs homologs are given at the end of the alignment.

Figure 2

Phylogenetic tree of Igf1r. The insulin receptor (InsR) served as the out-group. Bootstrap values are given, and the bar indicates number of substitutions per site. Accession numbers are after the organism. Igf1rs from different species are clustered together, indicating that generation of igf1r and insr took place in early vertebrate evolution.

Figure 3

Expression of igf1ra RNA and igf1rb RNA. (A,B) RT-PCR analysis of medaka igf1ra and igf1rb in adult tissues (A) and developing embryos (B). (C–F) Ovarian and testicular cryosections using antisense igf1ra and igf1rb probes and the signals were visualized by chromogenic staining. (G,H) qPCR results of igf1ra and igf1rb at different stages of follicles. I–V, stages of oocytes; sg, spermatogonia; sc, spermatocytes; st, spermatids; sm, sperm; gc, granulosa cells; tc, theca cells; se, Sertoli cells; le, Leydig cells. Scale bars, 100 µm.

Figure 4

Expression of igf1rb RNA and vasa RNA in the ovary. FISH on ovarian cryosections using antisense RNA probes and the signals were visualized by fluorescence staining. The vasa RNA was stained in red, and the igf1rb was stained in green. Nuclei were stained in blue with DAPI. (A,B) Different stages of oocytes (I–V), granulosa cells and theca cells were indicated by sticks. (C,D) The merges of vasa with igf1rb and vasa with igf1rb and DAPI. I–V, stages of oocytes; gc, granulosa cells; tc, theca cells. Scale bars, 25 µm.

Figure 5

Expression of igf1ra RNA and igf1rb RNA in the ovary. FISH on ovarian cryosections using antisense RNA probes and the signals were visualized by fluorescence staining. The igf1ra RNA was stained in red, and the igf1rb was stained in green. Nuclei were stained in blue with DAPI. (A,B) Different stages of oocytes (I–V), granulosa cells and theca cells were indicated by sticks. (C,D) Merges of igf1ra with igf1rb and igf1ra with igf1rb and DAPI. I–V, stages of oocytes; gc, granulosa cells; tc, theca cells. Scale bars, 25 µm.

Figure 6

Expression of vasa RNA and igf1rb RNA in the testis. After hybridization with antisense vasa and igf1rb RNA probes, the signals were visualized by fluorescence staining. Nuclei were stained blue by DAPI. (A–D) Merges of vasa with DAPI, igf1rb with DAPI, vasa with igf1rb, and vasa with igf1rb and DAPI. Sertoli cells were indicated by arrows. Vasa and igf1rb showed significantly different expression patterns. The vasa signal peaked in spermatogonia and then gradually decreased until it disappeared in sperm. Conversely, the igf1rb was obviously detected in spermatogonia, sperm, and Sertoli cells. sg, spermatogonia; sc, spermatocytes; st, spermatids; sm, sperm; se, Sertoli cells. Scale bars, 25 µm.

Figure 7

Expression of igf1ra RNA and igf1rb RNA in the testis. Adult testicular cryosections were hybridized to antisense RNA probes and the signals were visualized by fluorescence staining. Nuclei were stained blue by DAPI. (A,B) Lower magnification view showing different stages of spermatogenesis. Sertoli cells and Leydig cells were indicated by arrows. (C,D) Merges of igf1ra with igf1rb and igf1ra with igf1rb and DAPI. (E,F) Larger Magnification of panels D (white frame), highlighting the different cells. The igf1ra signal exists in sperm and Leydig cells. Notably, the igf1rb signal exists in spermatogonia, sperm, as well as Sertoli cells. se, Sertoli cells; le, Leydig cells; sm, sperm; sc, spermatocytes; sg, spermatogonia. Scale bars, 25 µm.