关键词: 山羊, 高通量测序, 卵泡发育, 颗粒细胞

Abstract:

【Background】 Dominant follicles (DF) and subordinate follicles (SF) were the most important two stages in the follicle development in the first follicle wave of goat. With further development of follicles, DF may eventually develop into mature follicles until ovulation, and SF will move towards atresia, while apoptosis of granulosa cells is the key factor leading to follicular atresia. However, the molecular mechanisms of promoting follicle dominance or causing its atresia are still unclear. 【Objective】This study was aimed to screen the key genes affecting follicular development and provide a theoretical basis for further exploring the regulation mechanism of follicular development by high-throughput sequencing of DF and SF granulosa cells in the first follicular wave of goat.【Method】Ten healthy Guizhou white goats were selected (1 year old), and the prostaglandin F_2α were injected respectively for estrus synchronization. B-type ultrasonography was used to detect the follicle growth situation, and then all of the goats were slaughtered when estrus had appeared for three days. DF (4.5-6 mm in diameter) and SF (3-4.5 mm in diameter) were obtained, granulosa cells were separated in the first follicle wave, respectively. Total RNA were extracted, and libraries were constructed and sequenced by Illumina Hiseq 2500 platform. FastQC was used to evaluate the quality of raw reads sequenced and filter them to obtain clean reads with high quality. Trinity was used to assemble clean reads from scratch to obtain unigenes. mRNA was obtained by comparing unigenes with goat RefSeq database using CLC Genomics Workbench. DESeq2 software was used to analyze the differential expression of the obtained mRNA. The goseq and kobas software were used for GO analysis and KEGG signal pathway analysis. Finally, qRT-PCR was used to verify the selected key genes that might affect the follicle development. 【Result】After the raw reads which obtained by sequencing were filtered, 43 217 934 clean reads were obtained from DF granulosa cells, accounting for 95.19% of raw reads. 40 766 348 clean reads were obtained from SF granulosa cells, accounting for 95.35% of raw reads. When the unigenes were compared with the RefSeq database of goat, a total of 33 896 annotated transcripts were obtained. Setting FPKM>1 and q value<0.05, a total of 13 644 genes were obtained in two types of follicle granulosa cells. By setting parameters: FPKM≥1, SF-FPKM/DF-FPKM>1, P<0.05, 695 differentially expressed mRNAs were obtained, of which 233 were significantly up-regulated and 462 were down-regulated in SF granulosa cells. GO functional enrichment analysis was performed on 695 differentially expressed mRNAs and concentrated in 42 groups of three major categories: biological processes accounted for 47.6%, cellular components 47.6%, and molecular functions 4.8%. KEGG signaling pathway analysis revealed 20 pathways, among which ribosome pathway related genes were most significantly enriched. Six genes that might be closely related to follicle development in goats were screened out, among which PRLR, PTX3 and RGN were up-regulated in subordinate follicles granulosa cells. DKK3, ALDH1A2 and RARRES1 were down-regulated. qRT-PCR showed that the expression trend of PRLR, RGN, DKK3, ALDH1A2 and RARRES1 was consistent with high-throughput sequencing results, and the expression level of RGN in SF granulosa cells was significantly higher than that of dominant follicles (P<0.01). The expression levels of DKK3, ALDH1A2 and RARRES1 in dominant follicles granulosa cells were significantly higher than subordinate follicles (P<0.01).【Conclusion】DKK3, ALDH1A2, RARRES1 and RGN had extremely significant differences in the expression levels of dominant follicles and subordinate follicles. It was speculated that those genes might promote the predominance of follicles or lead to atresia during the follicle development of goat, which was of great significance to further explore the regulation mechanism of follicular development.

Key words: goat, high-throughput sequencing, follicle development, granulosa cells