3.1 Expression Profile of Postnatal Ovarian Cycle Arrest and Normal Ovaries
To clarify the mechanism of postnatal ovarian cycle arrest, the transcriptome and proteome of estrus and anestrus ovaries were surveyed using RNA-Seq and iTRAQ techniques. A total of 18,036 genes were identified and quantified in the transcripts of ovary (Table 1). After filtering with FDR, 1,149 genes were found to be differentially expressed between estrus and anestrus ovaries, with 626 upregulated differentially expressed genes (DEGs) and 523 downregulated ones detected. A total of 56,530 transcripts were identified between estrus and anestrus ovaries with fold-changes greater than or equal to two and an FDR less than 0.01, where 2,294 differentially expressed transcripts (DETs) had 1,210 upregulated and 1,084 downregulated genes. A protein library was established by transcriptome results and differential protein analysis was carried out, where 4,457 proteins were identified. A total of 24 differentially expressed proteins (DEPs), of which 14 were upregulated and 10 were downregulated, were identified between estrus and anestrus ovaries with at least a 1.2-fold difference, Thirty-three transcriptome/protein pairs were synchronously upregulated. (Table S2).
Table 1
Overall features of the oestrus and anestrus ovaries expression profile.
Group name | Type | Number of genes | Number of proteins | Number of correlations |
Anestrus VS Oestru | Identification | 18036 | 4934 | 708 |
Anestrus VS Oestru | Quantitation | 18036 | 3889 | 487 |
Anestrus VS Oestru | Differential expression | 2294 | 24 | 3 |
3.2 Function Classification of DEGs and DEPs by COG
The COG database, an international standardized gene functional classification system was used to classify orthologous gene products. The upregulated and downregulated DEGs between postnatal estrus and anestrus ovaries were distributed in 21 and 17 COG categories, respectively. In Fig. 1, general function, the largest category in transcriptome, had 55 DEGs, 44 of which was upregulated. The upregulated DEPs between postnatal estrus and anestrus were distributed in translation, ribosomal structure and biogenesis, signal transduction mechanisms, posttranslational modification, protein turnover, chaperones and nucleotide transport and metabolism, all of which had one DEP. The downregulated DEPs were distributed in transcription and post-translational modification, protein turnover and chaperones, which had one DEP and two DEPs. The categories of cell cycle control, cell division, chromosome partitioning and cell wall, membrane and envelope biogenesis were directly associated with ovary growth.
3.3. Functional Classification of Enriched DEGs and DEPs by GO
The GO database was again used to classify the enriched DEGs and DEPs between postnatal estrus and anestrus ovaries. Based on sequence homology, they were categorized into three main categories as molecular function, biological process, and cellular component. A gene product might be associated with or located in one or more cellular components, so that it is active in one or more biological process, performing one or more molecular function. The DEGs were distributed in up to 7,495 GO terms and DEPs by contrast were classified into only 188 GO terms. The GO terms related to yak reproduction, environmental effect and rhythmic were selected. In the biological process category, the upregulated DEGs (Fig. 2A) and DEPs (Fig. 3A) were distributed in 51 and 26 GO categories, respectively. The terms of DEPs were all included in DEGs. In the category of biological process (Fig. 2A, Fig. 3A), the largest terms in transcripts and proteomics were cellular processes including 354 DEGs and cellular process with five DEPs. In respect to reproduction, a total of 75 DEGs were involved in reproduction and reproduction process. Contrastingly, no DEP was associated with the reproduction and reproduction process term. In respect of environmental effect, a total of 507 DEGs were associated with the terms of biological regulation and response to stimulus. Regarding proteomics, 30% of DEPs were associated with the terms of biological regulation and response to stimulus. Four DEGs were associated with the term of rhythmic process and no DEPs were associated. In biological process category (Fig. 2D, Fig. 3D), the downregulated DEGs and DEPs were distributed in 47 and 36 GO categories, respectively. In the category of biological process (Fig. 2D, Fig. 3D), the largest terms in transcripts and proteomics were cellular process with 187 DEGs and cellular process with seven DEPs. In respect to reproduction, a total of 43 DEGs and two DEPs were involved in the two terms of reproduction and reproduction process. In the aspect of environmental effect, a total of 284 DEGs were associated with the terms of biological regulation and response to stimulus. Regarding proteomics, 15.22% of DEPs were associated with the terms of biological regulation and response to stimulus. Three DEGs were associated with the term of rhythmic process and no DEPs were associated. In the cellular component category of upregulation (Fig. 2B, Fig. 3B), the term plasma membrane had the largest DEG number, occupying 14.56% of total annotated DEGs. Unlike DEGs, 25.64% of DEPs were distributed in the terms of cell and cell part which included 10 DEGs. Notably, most GO terms of proteomics were identical to those of transcripts. In the cellular component category of downregulation (Fig. 2E, Fig. 3E), the DEG number in the terms of cell part and cell were the largest, occupying 34.89% of total annotated DEGs. Like DEG, 25.64% of DEPs distributed in the terms of cell and cell part which included 14 DEG and most GO terms of proteomics were identical to those of transcripts. In the category of upregulated molecular function (Fig. 2C, Fig. 3C), most GO terms were associated with enzyme activity and binding. A total 247 DEGs fell into six GO terms having enzyme activities. In proteomics, two terms, including three DEPs, were related to enzyme activity. Thirty-five DEGs and three DGPs were involved the term of molecular function regulator. There were seven and six DEGs in the terms of electron carrier activity and antioxidant activity, respectively, but neither contained DEPs. Binding was the term that contained the largest DEG number at 305, which represented 50.33% of all DEGs annotated in GO terms. In the category of downregulated molecular function (Fig. 2F, Fig. 3F), most GO terms were also associated with enzyme activity and binding. A total of 158 DEGs fell into six GO terms having enzyme activities. In proteomics, four terms, including eight DEPs, were related to enzyme activity. Eleven DEGs and three DGPs were involved the term of molecular function regulator. There were four and two DEGs in the terms of electron carrier activity and antioxidant activity respectively, but neither contained DEP. Binding contained the largest DEG number at 165, which represented 47.97% of all DEGs annotated in GO terms.
3.4. Function Annotation of Enriched DEGs and DEPs by KEGG
The KEGG data base is a knowledge base for systematic analysis of gene functions that link genomic information with higher order functional information. Genes within the same pathway usually cooperate with each other to exercise their biological function, indicating that pathway-based analysis is a useful instrument for further understanding of the biological functions of genes. A KEGG analysis of DEGs and DEPs between postnatal estrus and anestrus ovaries was carried out. Genes and proteins involved in biochemical metabolism and signal transduction can be detected with pathway analysis and KEGG ways possessing DEGs and DEPs were evaluated in Fig. 4. The upregulated DEGs related to cellular processes had eight pathways and 15 DEGs (Fig. 4A), especially the pathways involved in reproductive processes, except p53 signaling and peroxisome. The DEPs were involved in reproductive processes except for focal adhesion (Fig. 4C). Six downregulated DEGs had two pathways associated with reproductive processes in endocytosis and phagosome, but no DEPs associated with reproduction (Fig. 4B-D). One upregulated DEG and two DEPs were in the cAMP signaling pathway and calcium signaling pathway associated with the reproduction of environmental information processing. In the category of upregulated DEGs organismal systems, progesterone oocyte maturation and the estrogen signaling pathway were involved in reproduction (Fig. 4A). Phototransduction and the GnRH signaling pathway were associated with the reproduction of DGPs. In the downregulated DEGs in organismal systems, two DEGs related to the estrogen signaling pathway were associated with reproduction, but not with DEPs associated with reproduction (Fig. 4B-D).
3.5 Validation of Selected DEGs and DEPs by PCR Analysis
To evaluate the gene expression profile, 25 genes including 22 upregulated genes and three downregulated genes based on the results of GO analysis belonged to response to stimulus, reproductive process, reproduction and rhythmic process were selected to undergo PCR (Table 2). Some of the selected genes were involved in reproduction directly, where ten genes in the two categories were all upregulated. In addition, some genes related to environmental effect were also chosen as response to stimulus related genes and they were also all upregulated. Rhythmic related genes were also examined where four genes were upregulated, and three genes were downregulated.
Table.2. Validation of selected DEGs by qRT-PCR analysis.
Gene ID
|
Gene product
|
qPCR
|
|
ONT
|
|
|
log2FC
|
regulated
|
log2FC
|
regulated
|
rhythmic process
|
|
|
|
|
gene16171
|
Lutropin-choriogonadotropic hormone receptor, partial [Bos mutus]
|
3.89
|
Up
|
3.95
|
Up
|
gene9978
|
PREDICTED: bone morphogenetic protein 15 [Bos mutus]
|
5.14
|
Up
|
5.11
|
Up
|
gene8885
|
Forkhead box protein R1
|
3.45
|
Up
|
3.43
|
Up
|
gene7394
|
Casein kinase I isoform alpha, partial [Bos mutus]
|
1.33
|
Up
|
1.23
|
Up
|
gene1350
|
Slit-like protein 3 protein, partial [Bos mutus]
|
-1.10
|
Down
|
-1.06
|
Down
|
gene533
|
hypothetical protein M91_01346 [Bos mutus]
|
-1.24
|
Down
|
-1.26
|
Down
|
gene1328
|
Inhibin beta A chain [Bos mutus]
|
-1.43
|
Down
|
-1.41
|
Down
|
reproductive process
|
|
|
|
|
gene21158
|
PREDICTED: placenta-specific protein 1 [Bos mutus]
|
2.36
|
Up
|
2.33
|
Up
|
gene17252
|
PREDICTED: cyclin-dependent kinases regulatory subunit 2 [Balaenoptera acutorostrata scammoni]
|
2.21
|
Up
|
2.15
|
Up
|
gene9481
|
PREDICTED: insulin-like 3 [Bos mutus]
|
2.25
|
Up
|
2.23
|
Up
|
gene9237
|
Zona pellucida sperm-binding protein 2 [Bos mutus]
|
4.67
|
Up
|
4.70
|
Up
|
gene7773
|
Calmegin, partial [Bos mutus]
|
1.77
|
Up
|
1.78
|
Up
|
reproduction
|
|
|
|
|
|
gene5675
|
Gap junction beta-5 protein [Bos mutus]
|
1.74
|
Up
|
1.75
|
Up
|
gene21158
|
PREDICTED: placenta-specific protein 1 [Bos mutus]
|
2.31
|
Up
|
2.33
|
Up
|
gene9210
|
PREDICTED: ubiquitin-conjugating enzyme E2 C isoform X1 [Bos mutus]
|
1.67
|
Up
|
1.62
|
Up
|
gene4775
|
PREDICTED: 15-hydroxyprostaglandin dehydrogenase [NAD(+)] isoform X1 [Bos mutus]
|
1.89
|
Up
|
1.95
|
Up
|
gene13254
|
oocyte-expressed protein homolog [Bos taurus]
|
3.22
|
Up
|
3.25
|
Up
|
response to stimulus
|
|
|
|
|
|
gene9034
|
Zinc finger protein 300, partial [Bos mutus]
|
2.33
|
Up
|
2.31
|
Up
|
gene14959
|
PREDICTED: placenta-specific gene 8 protein-like [Bos mutus]
|
1.44
|
Up
|
1.48
|
Up
|
gene14082
|
cell division control protein 2 homolog [Ovis aries]
|
1.65
|
Up
|
1.66
|
Up
|
gene9161
|
PREDICTED: securin isoform X1 [Bos mutus]
|
2.16
|
Up
|
2.17
|
Up
|
ONT.2585
|
Cleavage stimulation factor subunit 2 [Bos mutus]
|
1.67
|
Up
|
1.65
|
Up
|
gene6739
|
PCNA-associated factor, partial [Bos mutus]
|
1.89
|
Up
|
1.80
|
Up
|
gene4364
|
Macrophage-capping protein, partial [Bos mutus]
|
1.35
|
Up
|
1.31
|
Up
|
gene15253
|
Scavenger receptor class B member 1 [Bos mutus]
|
1.24
|
Up
|
1.21
|
Up
|
gene4783
|
Cyclin-A2, partial [Bos mutus]
|
1.92
|
Up
|
1.96
|
Up
|
gene3262
|
PREDICTED: growth/differentiation factor 9 [Bos mutus]
|
3.09
|
Up
|
3.01
|
Up
|
To sum up, the above results showed that the expression patterns of all selected genes as determined by PCR were in line with the results from the RNA-seq analysis, despite some differences in the expression levels. The results using samples from independent experiments at different reproductive stage indicated that the responses of ovary to hormone were reproducible. Therefore, the PCR results confirmed the reliability of results from RNA-seq based transcriptome analysis for identifying DEGs.