Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

Abstract

Bmpr2 plays a central role in the regulation of reproductive development in mammals, but its role during ovarian development in fish is still unclear. To ascertain the function of bmpr2 in ovarian development in the ricefield eel, we isolated and characterized the bmpr2 cDNA sequence; the localization of Bmpr2 protein was determined by immunohistochemical staining; and the expression patterns of bmpr2 in ovarian tissue incubated with FSH and hCG in vitro were analyzed. The full-length bmpr2 cDNA was 3311 bp, with 1061 amino acids encoded. Compared to other tissues, bmpr2 was abundantly expressed in the ovary and highly expressed in the early yolk accumulation (EV) stages of the ovary. In addition, a positive signal for Bmpr2 was detected in the cytoplasm of oocytes in primary growth (PG) and EV stages. In vitro, the expression level of gdf9, the ligand of bmpr2, in the 10 ng/mL FSH treatment group was significantly higher after incubation for 4 h than after incubation for different durations. However, bmpr2 expression in the 10 ng/mL FSH treatment group at 2 h, 4 h and 10 h was significantly lower. Importantly, the expression level of bmpr2 and gdf9 in the 100 IU/mL hCG group had similar changes that were significantly decreased at 4 h and 10 h. In summary, Bmpr2 might play a pivotal role in ovarian growth in the ricefield eel, and these results provide a better understanding of the function of bmpr2 in ovarian development and the basic data for further exploration of the regulatory mechanism of gdf9 in oocyte development.

Keywords: Bmpr2; FSH; Monopterus albus; hCG; immunoreactivity.

Figure 1

Nucleotide sequence information and amino acid sequence of the coding region of Bmpr2 in ricefield eel, Monopterus albus. The untranslated regions and translated regions are indicated by lowercase letters and uppercase letters, respectively. The predicted N-glycosylation sites are red boxes. The initiation codon (ATG) and stop codon (TAA) are marked in red color. Asterisks (*) indicate the translation stop codon.

Figure 2

Phylogenetic analysis and domain characteristics of Bmpr2 in ricefield eel, Monopterus albus. (A), The phylogenetic tree was researched by the neighbor-joining algorithm of Mega 11. The phylogeny was tested using the bootstrap method with 1000 replications. The numbers at nodes are bootstrap values (%). M. albus is marked in bold. The Bmpr2 protein sequences of vertebrates were obtained from Entrez (NCBI). (B), The characteristic Bmpr2 domains are conserved in ricefield eel orthologues. Bmpr2 is conservative in ligand-binding domain (ActRI/ActRII domain) and kinase domain, while the kinase domain is more conserved in Bmpr2. Numbers represent percentage identity of the predicted protein sequences with other type II BMP receptor orthologues (hBMPR2—Homo sapiens BMPR2, rBMPR2—Rattus BMPR2, gBMPR2—Gallus BMPR2, xBMPR2—Xenopus tropicalis BMPR2, mBmpr2—Micropterus salmoides Bmpr2b, sBmpr2—Scatophagus argus Bmpr2b, dBmpr2a—Danio rerio Bmpr2a, and dBmpr2b—Danio rerio Bmpr2b).

Figure 3

Expression of bmpr2 in different tissues of ricefield eel, Monopterus albus. (A), Relative mRNA levels of bmpr2 in tissues of ricefield eels were analyzed using RT-qPCR. (B), Bmpr2 immunoreactivity in the ovaries was evaluated using Western blot analysis. Anti-Bmpr2, primary antibody; Control, negative control; Bl, blood; Br, brain; Ey, eyes; He, heart; Ig, intersex gonads; In, intestines; Ki, kidneys; Li, liver; Mu, muscle; Ov, ovaries; Pi, pituitary; Sp, spleen; Te, testes. Results are expressed as means ± SEMs (n = 5). *, **, *** and **** are the significant differences of Duncan’s multiple comparisons, representing * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively.

Figure 4

Expression of bmpr2 in developing ovaries was analyzed in ricefield eel, Monopterus albus. (A), Expression of bmpr2 during ovarian development. (B), The localization of Bmpr2 immunoreactive signals (yellow and dot-like) in the ovary. (i), Primary growth stage; (ii), early vitellogenic stage; (iii), negative control. EV, early vitellogenic stage; MLV, middle to late vitellogenic stage; OM, oocyte in mature stage; OG, oogonium; PG, primary growth stage; PV, previtellogenic stage. Results are expressed as means ± SEMs (n = 5). * and **** are the significant differences in Duncan’s multiple comparisons, representing * p < 0.05 and **** p < 0.0001, respectively.

Figure 5

Immunofluorescence analysis of Bmpr2 and Gdf9 in the ovary of ricefield eel, Monopterus albus. Fluorescent images of Bmpr2 in the ovary captured by FITC: (A,a), DAPI; (B,b), FITC; (C), merged fluorescent images of (A,B). (c), Merged fluorescent images of (a,b). Fluorescent images of Gdf9 in the ovary captured by TRITC: (D,d), TRITC; (E), merged fluorescent images of (A,D); (e), merged fluorescent images of (a,d); (F), merged fluorescent images of (C,E); (f), merged fluorescent images of (c,e). PG, primary growth stage; PV, previtellogenic stage; EV, early vitellogenic stage. Immunofluorescence (green) shows Bmpr2 expression in the ovary. Immunofluorescence (orange-red signal) shows Gdf9 expression in the ovary. Nuclei are labeled with DAPI (blue). All photomicrographs were taken by an Olympus inverted research microscope.

Figure 6

Expression of gdf9 and bmpr2 in ovarian tissue of ricefield eel after FSH and hCG incubation in vitro. Results are expressed as means ± SEM (error bars), which were compared with the control group. (A,C) The relative expression level of gdf9 and bmpr2 after FSH incubation; (B,D), the relative expression level of gdf9 and bmpr2 after hCG incubation. FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; IU, international unit. *, **, *** and **** are the significant differences in Duncan’s multiple comparisons, representing * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively.