Transcriptome Analysis Provides Insights into Copulation, Fertilization, and Gestation in Sebastes schlegelii

Abstract

Among the viviparous marine teleosts of China, the black rockfish (Sebastes schlegelii Hilgendorf) is one of the most economically important. In addition to copulation and internal fertilization, it features lengthy sperm storage in the female ovary as well as a high rate of abortion. A network of gene regulation is necessary for these processes. To elucidate the mechanisms of copulation, fertilization, and gestation, it is essential to determine the genetic basis of viviparous teleost oogenesis and embryogenesis. In this study, we analyzed the transcriptome of the ovary during different developmental phases to investigate the dynamic changes that occur. We constructed 24 ovary transcriptomes. In order to investigate the regulation of embryogenesis, differentially expressed genes (DEGs) with specific expression patterns were subjected to gene ontology annotation, pathway analyses, and weighted gene co-expression network analysis (WGCNA). The up-regulated genes were significantly enriched in focal adhesion, regulation of the actin cytoskeleton, Wnt, and ECM-receptor interaction signaling pathways. As a result of our study, we provide omics evidence for copulation, fertilization, and gestation in viviparous marine teleosts. Decoding the S. schlegelii gene regulation network, as well as providing new insights into embryogenesis, is highly valuable to researchers in the marine teleost reproduction sciences.

Keywords: RNA-seq; S. schlegelii; copulation; embryogenesis; fertilization.

Figure 1

The pattern of the adult S. schlegelii Reproductive cycle. The male of S. schlegelii was mature and copulated from the middle of October to December. The female of S. schlegelii was maturation at about April. The fertilization in vivo occurred between April and May. The time was closely related to the water temperature. Aug–Oct: spermatogenesis; Oct–Dec: sperm maturation; Nov: copulation; Oct–Mar: vitellogenesis; Apr: fertilization; May–Jun: gestation. Mar: March; Apr: April; May: May; Jun: June; Aug: August; Oct: October; Nov: November; Dec: December.

Figure 2

The characteristics of the ovary during the different developmental stages. (A) II stage, (B) III stage, (C) IIIIV stage, (D) V_1 stage, (E) V_2 stage, (F) bla stage, (G) gas stage, (H) lab stage, (I) VI stage, (J) high resolution of I image (scalebar, 100 μm). At the MII stage, the ovary shape was cylindrical, and its color was pink (A1). The cell type was mainly the oogonium and early peri-nucleolus (A). At the MIII stage, the ovary volume expanded, and the color was light yellow (B1). The cell types were mainly late peri-nucleolus oocytes (B). At the IIIIV stage, the yolk granules of oocytes constantly accumulated (C), and the ovary volume was expanded (C1). At the V_1 stage, the ovary matured and was rich with blood vessels (D1). The oocyte was filled with yolk granules, and lipid droplet vesicles were scattered (D). At the V_2 stage, the embryo was in the cleavage stage after fertilization (E,E1). At the bla stage, the embryo was in the blastula stage (F,F1). At the gas stage, embryos are in the gastrulation stage (G,G1). At the lab stage, the embryo is in the labor stage (H,H1). At this stage, the embryos have completed the hatching process and are born. The eyes were particularly obvious, and the ovaries appeared black. At the VI stage, the ovary shrank (I1), the cell type was mainly oogonia and early peri-nucleolus stage, and there was much more connective tissue, similar to that in oviparous teleosts after ovulation (I). J is the high resolution of I image (scalebar, 100 μm). OW: ovary wall; OGA, oogonia; EP, early peri-nucleolus stage oocyte; LP, late peri-nucleolus stage oocyte; ODS, oocytes at oil droplet stage; PYG, primary yolk globule stage; TYG, oocytes at tertiary yolk globule stage; FP, follicular placenta; BV, blood vessel; BLAE, bla stage embryo; GASE, gas stage embryo.

Figure 3

DEGs between the different ovarian developmental stages. (A) Summary of DEGs. The x-axis represents compared samples. The y-axis represents DEG numbers. The blue colour represents upregulated DEGs and the orange colour represents downregulated DEGs. (B) Venn diagram of ovarian transcripts from different reproductive phases.

Figure 4

The heatmap of reproduction related DEGs expression pattern at different reproductive phases. The red and blue colors indicated up- and down-regulated transcripts, respectively.

Figure 5

Gene ontology (GO) analysis of the DEG(s) with a two-fold difference. (A) IIIIV vs II, (B) IIIIV vs III, (C) V_1 vs III, (D) V_2 vs IIIIV, (E) gas vs lab. The x-axis shows the number of genes in each term. The y-axis shows the specific terms. The asterisk represents the corrected p value < 0.05 for each GO term.

Figure 6

Scatter plots of enriched KEGG pathways for DEG(s) with a two-fold difference from the comparison of different stages. (A) IIIIV vs II, (B) IIIIV vs III, (C) V_1 vs III, (D) V_2 vs IIIIV, (E) gas vs lab. The rich factor is the ratio of the number of DEGs for a certain KEGG over the total genes in that pathway. Q value is the p value after correction for multiple testing. The color and size of the circles are q values and DEGs numbers, respectively.

Figure 7

Validation of the DEGs by RT-qPCR. The expression levels of cyp11a1, star, lhcgr, hsd17b3, itr, and cxcl12 during different developmental stages were detected by RT-qPCR. For reference genes, 18S were used for normalization of RT-qPCR data. Bars represent the standard deviation (SD). The x-axis indicates the developmental stage. The y-axis shows the relative expression level of genes. A: cyp11a1; B: star; C: lhcgr; D: hsd17b3; E: itr; F: cxcl12.

Figure 8

WGCNA analysis of RNA-seq and network visualization. (A): The heatmap of module-gene correlation. Each row and column represent a gene, and the darker the color of each point represents the stronger connectivity between the two genes. (B): The eigengene dendrogram of module membership. (C): Key genes related to copulation of the skyblue module. (D): The key genes related to fertilization of the blue module. (E): The key genes related to gestation of the green module. (F) The key genes related to the maturation and fertilization of purple modules. Note: The module was selected according to the relationship between the module and samples’ expression heatmap. (C–F) was a visualization by Cystoscope. The gene connectivity in each module was sorted by the connectivity value. The high connectivity genes were selected (the threshold value according to the gene numbers).