1. Well-developed vascular system in the ovary
The ovary structure of black rockfish was analyzed for developmental series, from post-mating to post-parturition. The exterior and interior ovarian blood vessels were observed both by eye and microscope after dissection (Fig.1). Exterior observations showed that a main vessel is located in the middle of ovary wall. Branch vessels and more capillaries extended into the ovary (Fig.1A-C). Microscopic observations showed that the capillaries spread over the ovary. It is important to note that more interior vessels were observed with the process of final oocyte maturation and gestation (Fig.1D-H). Highly vascular membranous tissue was found adjacent to the egg membrane, especially in the stages of fertilization and gestation (Fig.1F-H). Many genes involved in angiogenesis or vasoconstriction had a relatively higher expression in ovarian wall or eggs compared to other genes, which provided the evidence of new vascular formation from molecular level. (Fig. S1 and Fig. S2). In addition, it is interesting to note that three of five vasoconstriction regulating genes in ovarian wall were Bdkrb2 genes (Fig. S2).
2. Characterization of Bdkrb2 genes in black rockfish
A total of eight Bdkrb2 genes were identified in black rockfish genome, including Ssc_10023113, Ssc_10023114, Ssc_10023115, Ssc_10023116, Ssc_10023117, Ssc_10023118, Ssc_10023119 and Ssc_10023120. They were located on chromosome 14, which were arranged in a tandem array, forming a gene cluster spanning about 50 kb (Fig. 2A). Amino acid alignments of Bdkrb2 genes in black rockfish revealed that Ssc_10023116 was highly homologous to Ssc_10023117 (82%), Ssc_10023118 (84%), Ssc_10023119 (81%) and Ssc_10023120 (78%) (Table. S1). Whereas the similarity among Ssc_10023113, Ssc_10023114 and Ssc_2310023115 was only around 35% (Fig. S3, Fig. S4 and Table. S1). The open reading frame (ORF) of Ssc_10023113 was 1092 bp long, encoding 363 amino acids. The ORF of Ssc_10023114 is 1110 bp long, encoding 369 amino acids. The ORF of Ssc_10023115 was 1029 bp long, encoding 342 amino acids. Each one of the BDKRB2 proteins encoded by Ssc_10023113, Ssc_10023114 and Ssc_10023115 contained seven transmembrane (TM) domains, forming a typical G protein (Fig. 2B). The ORF of Ssc_10023120 was 858 bp long, encoding 285 amino acid. The rest four genes, Ssc_10023116, Ssc_10023117, Ssc_10023118, and Ssc_10023119, each contained a 900 bp long ORF and encoded 299 amino acids. Each one of the BDKRB2 proteins encoded by Ssc_10023116, Ssc_10023117, Ssc_10023118, Ssc_10023119 and Ssc_10023120 contained only six TM domains, resulting in protein conformational changes (Fig. 2B, 2C). Changes were detected by 3D structure prediction. The proteins with seven TM domains had both intracellular and extracellular terminus region, however, the proteins with six TM domains only had intracellular terminus region (Fig. 2C). The conformational changes may cause functional changes.
3. Phylogenetic relationship among Bdkrb2 genes
The phylogenetic tree showed that the BDKRB2 orthologs of teleosts were clustered into a clade. It's worth noticing that Ssc_10023113, which had seven TM domains, clustered into a sub-clade with all of the Bdkrb2 genes only owning six TM domains, including Ssc_10023116, Ssc_10023117, Ssc_10023118, Ssc_10023119 and Ssc_10023120. The remaining two of Bdkrb2 genes, Ssc_10023114 and Ssc_10023115, which also had seven complete TM domains, were clustered into another sub-clade.
4. Expression and preliminary function analysis of Bdkrb2 genes
To understand the functional differentiation of Bdkrb2 genes in black rockfish, the expression pattern was analyzed in different tissues. The result showed that eight Bdkrb2 genes can be roughly classified into three patterns (Fig. 4). Ssc_10023113 and Ssc_10023119 were highly expressed in gills. Ssc_10023114, Ssc_10023115 and Ssc_10023116 were highly expressed in intestines, regardless of sex. Ssc_10023117, Ssc_10023118 and Ssc_10023120 were highly expressed in the ovarian wall in females and genitalia in males (Fig. 4A). Among the eight genes, Ssc_10023113 had the widest tissue expression, including the brain, gill, intestine, ovarian wall and genitalia. Expressions of Bdkrb2 genes in the ovarian wall were analyzed in detail. Ssc_10023117 and Ssc_10023118 had a significant higher expression in the tissue of ovarian wall at the stage of post_mating and pre_ fertilization (Fig. 4B).
To explore the function of Bdkrb2 genes, Ssc_10023113 and Ssc_10023117 was chosen as the representatives of genes, which encoded BDKRB2 protein with seven and six transmembrane domains, respectively. Capped mRNA of two genes were synthesized in vitro. Transgenic zebrafish strain (Fli1a: EGFP) was used for functional study due to technical limitations in the black rockfish. Both overexpression of Ssc_10023113 and Ssc_10023117 resulted in different degrees of malformation in 24 h post-fertilization (hpf) embryos, including rostral-caudal axis, edema of the pericardial cavity (Fig. 5A). The rates of deformation were 55.77% and 35.65% for Ssc_10023113 and Ssc_10023117 genes, respectively, showing no statistical significance by chi-square test (P=0.368). Confocal microscopy observation showed that overexpression of Ssc_10023113 and Ssc_10023117 both resulted in abnormal vascular development, including increased diameter of the common cardinal veins (CCVs), anomalous and undifferentiated intersegmental vessel (Se). It indicates that Ssc_10023113 and Ssc_10023117 had similar function in regulation of embryo development and vascular formation.
5. Bdkrb2 genes play a role in the ovary wall in the reproductive cycle
To investigate the function of Bdkrb2 genes in the ovary in the reproductive cycle, samples of connective tissue rich in blood vessels covering the egg membrane, embryos and ovarian wall at different stages were collected for RNA-seq. The expression analysis provided more detailed information about the different regulatory pattern of angiogenesis and vasoconstriction in the three kinds of samples (Fig. S5 and Fig. S6). Angiogenesis and vasoconstriction related genes have continuous expression at pre-fertilization and gestation. All Bdkrb2 genes are expressed in the ovarian wall, however, their expression periods were not similar (Fig. 6). Jonckheere-Terpstra Test was performed to demonstrate that mean expression of selected gene differed among the developmental stages. The result showed that the expression level of Ssc_10023113 (P=0.006), Ssc_10023114 (P=0.015), Ssc_10023116 (P=0.000), Ssc_10023117 (P=0.001), Ssc_10023118 (P=0.001) or Ssc_10023119 (P=0.038) was significantly different among the 20 stages sampled. Ssc_10023113, Ssc_10023116, Ssc_10023117, Ssc_10023118, Ssc_10023119 and Ssc_10023120 had an expression at the stage of pre-fertilization and pre-hatching, suggesting that these genes play a role in preparing for fertilization and hatching. Furthermore, Ssc_10023113, Ssc_10023114 and Ssc_10023115 also highly expressed at the stages at the stage of hatching, suggesting that these genes play a role in hatching.