Comparative analysis of ovarian transcriptomes between prolific and non-prolific goat breeds via high-throughput sequencing.

To increase the current understanding of the gene expression in the pre-ovulatory ovary and identify the key genes involved in the regulation of ovulation rate, we compared the transcriptomes of ovaries from the prolific Jintang black goat (JTG) and the non-prolific Tibetan goat (TBG) during the follicular phase using the Illumina RNA-Seq method. Three ovarian libraries were constructed for each breed. On average, we obtained approximately 49.2 and 45.9 million reads for each individual ovary of TBGs and JTGs, respectively, of which 79.76% and 78.67% reads were covered in the genome database. A total of 407 differentially expressed genes (DEG) were detected between these two breeds, in which 316 were upregulated, and 91 were downregulated in the ovaries of JTGs versus TBGs. Based on the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, some of these DEGs potentially play an important role in controlling the development of ovarian follicles. SRD5A2, MSMB, STAR and 3BHSD, etc. were the most significantly differentially expressed between these two distinct breeds. In addition, each ovary expressed 1,066 versus 989 novel transcripts, and 171,829 versus 140,529 putative SNPs in TBGs versus JTBs, respectively. All data sets (GEO and dbSNP) were available via public repositories. Our study provides insight into the transcriptional regulation of the ovaries of two distinct breeds of goats that might serve as a key resource for understanding goat fecundity. SRD5A2, MSMB, STAR and 3BHSD may be associated with the high fecundity of JTGs.

Characterization of transcriptional complexity during pre-implantation development of the yak (Bos grunniens) using RNA-Seq.

The objective of this study was to investigate the mechanism that regulates pre-implantation development of the yak (Bos grunniens). We determined the transcriptomes of in vitro-produced yak embryos at two-cell, four-cell, eight-cell stages, and morula and blastocyst using the Illumina RNA-seq for the first time. We obtained 47.36-50.86 million clean reads for each stage, of which, 85.65%-90.02% reads were covered in the reference genome. A total of 17,368 genes were expressed during the two-cell stage to blastocyst of the yak, of which 7,236 genes were co-expressed at all stages, whereas 10,132 genes were stage-specific expression. Transcripts from 9,827 to 14,893 different genes were detected in various developmental stages. When |log2 ratio| ≥ 1 and q-value <0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 6,922-10,555 DEGs between any two consecutive stages. The GO distributions of these DEGs were classified into three categories: biological processes (23 terms), cellular components (22 terms) and molecular functions (22 terms). Pathway analysis revealed 310 pathways of the DEGs that were operative in early pre-implantation yak development, of which 32 were the significantly enriched pathways. In conclusion, this is the first report to investigate the mechanism that regulates yak embryonic development using high-throughput sequencing, which provides a comprehensive framework of transcriptome landscapes of yak pre-implantation embryos.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.

Cloning and expression analysis of the follicle-stimulating hormone receptor (FSHR) gene in the reproductive axis of female yaks (Bos grunniens).

Follicle-stimulating hormone receptor (FSHR) plays a central role in promoting follicle maturation through the follicle-stimulating hormone (FSH)-mediated cAMP pathway in animals. The objectives of the present study were to clone the FSHR gene of yaks (Bos grunniens) and compare differences in FSHR mRNA expression in the reproductive axis between yaks and cattle. Hypothalamus, anterior pituitary, oviduct, ovary, and uterus tissue samples were collected from adult female yaks (n = 5) and cattle (n = 5) during the follicular phase. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we found that the FSHR coding region of the yak is 2088 bp and encodes 695 amino acids. Its amino acid sequence showed 99.38%-72.22% similarity to the homologous genes of cattle, goats, sheep, cats, donkeys, horses, humans, chickens, monkeys, mice, rats, and wild boar. Real-time PCR analysis revealed that the FSHR gene was expressed in all tissues examined. Expression of the FSHR gene in the yak was higher in the uterus than other tissues (P < 0.05) but, in cattle, was higher in the ovary than other tissues (P < 0.05). The FSHR gene expression level in the cattle ovary was significantly higher than that in the yak ovary (P < 0.01). These results indicate that the FSHR gene is relatively conserved in the course of animal evolution. The variation in sequence and expression level of FSHR between the two species might be associated with the difference in their reproduction.

Identification of differential abundances of mRNA transcript in cumulus cells and CCND1 associated with yak oocyte developmental competence.

The development of an accurate and noninvasive preselection process for competent oocytes is essential to achieve a highly efficient in vitro production (IVP) of embryos. Cumulus cells (CCs) have important functions in oocyte growth, development, maturation, and fertilization. It, therefore, is important to know if the quality of oocytes can be ascertained by assessment of gene expression of the surrounding CCs or not. The aim of this study was to identify differentially expressed genes in yak CCs from oocytes with varying developmental competences as possible biomarkers for distinguishing oocyte competence. The isolated CCs were pooled into immature and mature groups in accordance with the maturation outcome of oocytes. A total of 9516 genes were differentially expressed in the two CC categories (P <  0.05). With a minimum change of 2.5-fold, 45 up-regulated and 79 down-regulated genes were observed in CCs belonging to the mature group compared with those in the immature group (P <  0.01). These genes were primarily enriched for the cell cycle, meiosis, cell signaling, metabolism, and apoptosis. The selected candidate genes (CCND1, BMP15, GDF9, H19, KLF4, GPC1, SYCP3, and CTSB) were validated using quantitative real-time polymerase chain reaction (RT-qPCR) and there were expression patterns similar to those detected with transcriptome analysis. The CCs from fertilized oocytes arrested at the 2-cell (2-cell group), or 8-cell (8-cell group) stages or that developed into blastocysts (the blastocyst group) had a 1.5-, 1.8-, and 2.3-fold increase, respectively, in mRNA relative abundance of CCND1 compared with CCs from unfertilized oocytes (P <  0.05). The results with the RT-qPCR analysis confirmed that the relative abundance of CCND1 mRNA in CCs was associated with oocyte developmental competence. In conclusion, RNA-Seq is useful in extracting transcriptomes and selecting markers associated with oocyte developmental competence. Furthermore, the expression of the CCND1 gene in yak CCs can be used to preselect oocytes for IVP efficiency.