The exploration of miRNAs and mRNAs profiles revealed the molecular mechanisms of cattle-yak male infertility

doi:10.21203/rs.3.rs-1616376/v1

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The exploration of miRNAs and mRNAs profiles revealed the molecular mechanisms of cattle-yak male infertility

https://doi.org/10.21203/rs.3.rs-1616376/v1

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Background

Cattle-yak, the first-generation offspring of cattle and yak, inherited many excellent characteristics from their parents. However, F1 male hybrid infertility restricts the utilization of heterosis greatly. The coding genes can transcript into such mRNAs and further translate into proteins to maintain the activities of organisms. MiRNAs are endogenous small noncoding RNA that silenced their target mRNA expression by high complementary binding sites. In recent years, mRNA and miRNA profiles produced by high-throughput sequencer had interpreted the molecular mechanisms of numerous biological phenomena. However, based on the cattle-yak male sterility, the comprehensive comparison of testicular tissue RNA profiles of cattle, yak and cattle-yak have not been conducted.

Results

Testicular histology indicated that the seminiferous tubules became vacuolated and there are few active germs cells can be seen. With the RNA-seq analysis we finally identified 47 up-regulated miRNAs and 34 down-regulated miRNAs in cattle, 16 up-regulated miRNAs and 21 down-regulated miRNAs in cattle and yaks compared with cattle-yak, respectively. Furthermore, we found that the known miRNA bta-miR-7 was commonly down-regulated in cattle-yak group, revealed the vital role of miR-7 in the regulation of reproductive processes. With the target prediction, the coding genes MYRFL, FANCA, INSL3, USP9X, SHF became the regulated candidates. The network analysis of GO to GO and pathway to pathway annotated such hub terms and genes related to many regulatory functions. We also performed GSE GO and GSE KEGG to display the function of DE gene sets authenticated in our previous study and found that the gene sets most enriched in ATP binding, DNA binding, reproduction process and MAPK signaling pathway, uncovered the relevance between the DE genes and cattle-yak male infertility.

Conclusions

We first obtained the compared miRNAs profiles of testicular tissue samples among cattle, yak and cattle-yak and analyzed the fundamental functions of their target genes. As a complement, GSEA of mRNA profiles identified in our previous study was also be conducted. This study preliminarily revealed some miRNAs related to reproductive processes such as bta-miR-7 and such critical regulatory pathways. Our research is of great significance to explore the molecular mechanism of cattle-yak infertility and look for strategies to resolve this difficult problem.

cattle-yak

miRNA

mRNA

RNA-seq

bta-miR-7

male sterility

molecular mechanisms

Studies have revealed that only about 2% genome transcripts have protein-coding ability[1]. However, as the most numerous components of genomic transcriptional products, the noncoding RNA has also been confirmed that play significant roles in regulating gene expression in some different levels such as transcription, RNA processing, translation and epigenetic regulation[2–4].The noncoding RNAs can be classified into small and long ones based on length[5]. MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules 19 to 25 nucleotides in size that regulate posttranscriptional silencing of target genes, and a single miRNA can influence the expression patterns of many genes through targeting hundreds of mRNAs[6]. In mammals, there are many studies that focus on miRNAs and increasing evidences had demonstrated that the miRNAs can participate in multiple biological processes such as proliferation[7], differentiation[8, 9] and apoptosis[10]. Moreover, miRNAs can act as epigenetic modulators to affect the protein levels of the target mRNAs without modifying the gene sequences, and the expression of miRNAs is also regulated by epigenetic machinery such as DNA methylation, RNA modification[11].MiRNAs were also been validated that participate in processes of dyszoospermia and male infertility[12].

Cattle-yak, an excellent hybrid of cattle (Bos taurus) and yak (Bos grunniens), exhibit more excellent economic traits than its parents such as meat quality, cold resistance and growth performance. For example, the phospholipids containing long-chain polyunsaturated fatty acids has been confirmed that are more abundant in the muscles of plateau cattle (cattle-yak and yak) than plain cattle (cattle), which are considered beneficial to human health [13–15]. But the Haldane’s rule demonstrated that the heterogametic sex of first generation between hybrids is interfile. Male infertility of F1 generation of cattle-yak resulted from spermatogenic arrest has greatly restricted the effective utilization of heterosis from the hybrids and the cultivation of new breeds[16, 17]. Male cattle-yak has normal external genitalia, libido and mating behavior, but testicular histology revealed that the seminiferous tubule walls of cattle-yak was thinner and spermatogenesis was arrested at the stage of spermatogonia and primary, which was hardly ever able to proceed further than meiosis I[18]. In epigenetic, different with yak, the expression levels of acetyl-histone H3 Lys9 (ACK9) in cattle-yak was abnormal and the 5-methylcytosine level was higher[19]. Studies also observed that the decreased expression level of androgens and androgen receptor increased expression of 3β-hydroxysteroid dehydrogenase(3βHSD) in the Leydig cells of cattle-yak hybrid testes[20]. As a member of deleted azoospermia family genes, Boule gene was also be proved that play important role in regulating primordial germ-cell formation [21].Entirely different with cattle, the expression level of Boule gene was lower because the long CpG island in the promoter was hypermethylated in the testes of cattle-yak hybrids, and the differences in the methylation level between cattle and cattle-yak hybrids were larger in the core promoter than outside the core promoter[22].With the development of molecular biology in recent decades, such genes and transcription factors associated with male infertility had been identified. For example, DNA-binding protein PRDM9 is related to double strand breaks initiating meiotic recombination and the position of DSBs is closely related to the fertility in male hybrids[23]. Researchers had found that the expression level of bovine meiosis defective 1 (bMei1) involved in spermatogenesis was significantly decreased and methylation level of promoter and gene body in the testis of cattle–yak was significantly increased compared with cattle. On the contrary, with the treatment of methyltransferase inhibitor 5-aza-2′ deoxycytidine (5-Aza-CdR), the activation of bMei1 would recover, further confirms that the bMei1 expression level is associated with methylation[24]. Besides, as a subfamily of Argonaute protein family, PIWI proteins are associated with a novel class of small RNAs known as PIWI-interacting RNAs and PIWIL1 mRNA expression in the testes of cattle-yaks is significantly lower than that of cattle or yaks because of higher methylation levels of PIWIL1 gene[25, 26].Rad51 and meiotic specific DMC1 recombinases can act synergistically in the process of homologous recombination, study revealed that Rad51 and DMC1 are extremely low in the male cattle-yak testis with a corresponding higher incidence of germ cell apoptosis, highlighting the important role of Rad51 gene in regulating spermatogenesis[27].As a family of transcription factors, heat shock factors can regulate the expression levels of heat shock proteins (HSP) and perform crucial roles during gametogenesis and development in physiological conditions, and study has demonstrated that infertility of cattle-yak may be caused by the upregulation of HSP53[28–30]. With the development of high-throughput sequencing technology, many studies had preliminary performed comparative transcriptome profiles analysis in testes tissues and epididymis of cattle-yak and yak [31–34]. However, there are few studies that comprehensively analyzed the RNA profiles of cattle, yak and cattle-yak testicular tissues.

In our previous study, we had compared the testicular tissues lncRNA and mRNA profiles differential expression of cattle, yaks and cattle-yak[35]. Similarly, we also utilized the next generation sequencing technology to conduct the comparison of three varieties miRNA profiles. In addition, we performed GSEA of DE gene sets to identify the differences of gene expression patterns. We hope to have a deeper understanding of the molecular mechanism of male cattle-yak infertility.

Testicular tissues histological characteristics of nine samples

Compared with cattle and yaks, the testicular tissues sections of cattle-yak showed that seminiferous tubules were highly vacuolated and the walls became thinner. Opposite with cattle and yak(Fig.1 A,B), the area of the longitudinal section of testis were expanded and there are few germ cells or spermatocytes can be seen in the seminiferous tubules of cattle-yak (Fig.1 C).

Quality control of sequencing data

The raw data of fastq format was removed 3’ adaptors with cutadapt under specific settings parameters. Then the trimmomatic software was used to filter the low-quality reads and clean reads were converted to fasta format. There were 3,192,697 to 6,617,422 unique clean reads generated with filtering the low-quality reads and covering the repeated reads. The GC percentage of these reads was from 46.31% to 49.29%, and all the Q30 values of these reads were surpass to 96%. All the clean data of each sample can be used to reads mapping and further analysis (Table 1).

Table 1. The statistics results of clean data

Sample	Reads count	Unique count	Bases count	Average length	Q20	Q30	GC percentage
h1	12,855,276	3,351,413	360,561,690	28.05	97.87%	96.54%	47.02%
h2	12,886,450	3,783,343	367,250,682	28.5	97.78%	96.38%	46.35%
h3	15,070,070	4,342,944	424,855,422	28.19	97.76%	96.34%	46.31%
m1	10,694,999	3,881,169	278,614,344	26.05	97.78%	96.40%	48.41%
m2	12,384,809	4,380,294	331,397,620	26.76	97.68%	96.22%	47.51%
m3	14,831,950	5,470,038	382,097,489	25.76	97.72%	96.32%	47.80%
p1	13,443,245	4,640,237	347,901,405	25.88	97.84%	96.52%	48.62%
p2	14,979,305	6,617,422	392,403,979	26.2	97.80%	96.44%	48.67%
p3	12,329,720	3,192,697	317,680,881	25.77	97.82%	96.47%	49.29%

Reads mapping and filtration

Clean reads were mapped to the bovine reference genome, exon sequence and intron sequence. The reads that can be mapped to the reference genome and cannot be mapped to the exon sequence were persevered. In addition, blastn was used to aligned the reads to the Rfam database (rRNA, snRNA, snoRNA, tRNA) and then filtered the mapped reads (Supplementary table1).

The miRNA family and expression level analysis

After filtering the reads that did not belong to miRNAs, the mirDeep2 was used to predict the novel miRNA and secondary structure of miRNAs. After quantification of miRNAs, we analyzed the miRNA family to further understand the function of miRNAs to their target genes (Fig 2). Then these counts were normalized with RPM. The RPM distribution of nine samples can be seen as the RPM boxplot (Fig 3. a). At the same time, we analysis the principal component of nine samples and the cattle, yak and cattle-yak can be divided into H, M and P groups (Fig 3. b). With the miRNA expression levels of each sample, we further analyzed the correlation among three samples in one group and the distance among three groups, the samples of each group were highly relevant and the distance among three groups was obvious (Fig 4).

Differential expression analysis of miRNA among nine samples

The R package edgeR was used to analyze the differential miRNA expression among nine samples with CPM >1, p-value < 0.05, |log₂FoldChange|>1. And then the clustering analysis and differentially expression of H vs P and M vs P were then pictured with heatmap (Fig 5). There were 81(47 up-regulated and 34 down-regulated) and 37(16 up-regulated and 21 down-regulated) differentially expressed miRNAs in H vs P and M vs P, respectively (Fig 6). The list of differentially expressed miRNAs can be seen as Supplementary table2. Then, we consolidate the common up-regulated and down-regulated miRNAs of cattle-yak in the merged analysis of H vs P and M vs P (Fig 7). We plan to study the function of common miRNAs to their target genes next step and further understand the mechanism of male sterility.

Functional enrichment and network analysis

With the list of the differentially expressed miRNA of H vs P and M vs P, we use miRanda software to predict their target genes. In H vs P group, there are 6 up-regulated miRNAs and 1 down-regulated miRNA in cattle can be predicted their target genes (Table 2). Besides, M vs P group filtered 2 up-regulated and 3 down-regulated miRNA in yak can target the protein coding genes and further regulate the associated signaling pathways (Table 3). We also observed that bta-miR-7 is commonly down regulated in cattle-yak between H vs P and M vs P, the predicted target genes MYRFL, FANCA, INSL3, USP9X, SHF may play decisive roles in leading to the male-sterility of cattle-yak (Fig 7). After target prediction, we carried out GO and KEGG enrichment analysis to understand the function of the predicted coding genes. With three aspects (molecular function, biological process, cellular component) of GO enrichment, we selected top 10 GO terms to describe the function of these genes, we found that the up-regulated genes of cattle most engaged in the negative regulation of ubiquitin-dependent protein catabolic process (GO:2000059), positive regulation of cell migration (GO:0030335), thiol−dependent ubiquitinyl hydrolase activity (GO:0036459) and positive regulation of mitochondrial DNA replication (GO:0090297). Besides, KEGG enrichment analysis demonstrated that these target genes most enriched in cell cycle (bta04110), RNA transport(bta03013), PI3K−Akt signaling pathway(bta04151), Focal adhesion (bta04510) pathways. On the other hand, the up-regulated genes in yak are most enriched in the DNA-binding transcription factor activity (GO:0003700), positive regulation of DNA demethylation (GO:1901537), protein deubiquitination (GO:0016579), zinc ion binding (GO:0008270) terms and relaxin signaling pathway (bta04926), RNA polymerase (bta03020) pathways, such the up-regulated GO terms and pathways in cattle and yak may positively participate in the reproductive processes (Fig 8. A-D). With the analysis of miR-7 target genes function annotation, we found these genes enriched in hormone activity, signaling receptor binding and activator activity, molecular function regulator process, Fanconi anemia pathway and relaxing signaling pathway that associated with hormone secretion and signal transduction (Fig 8. E-F). At the same time, we performed Gene Set Enrichment Analysis (GSEA) to annotate and interpret enrichment results of set of DEGs, in order to characterize the function of DEGs more accurately. We found that the gene sets of cattle enriched in the ATP binding, reproduction and developmental process involved in reproduction processes and focal adhesion, olfactory transduction and MAPK signaling pathway in H vs P group. Likewise, the gene sets of yaks are most involved in the ATP binding, DNA binding and reproduction processes, as well as the cytokine receptor interaction and MAPK signaling pathways (Fig 9.) We also found that the positive regulation of cell migration (GO:0030335), negative regulation of cell adhesion (GO:0007162), SMAD2-SMAD3 protein complex processes, PIK3R1 gene, SMAD2 gene, relaxion signaling pathway (bta-04926) and cellular senescence (bta04218) were located in the center of GO and KEGG regulatory network of H vs P DE miRNAs target genes (Fig.10 A-C). Similarly, in the M vs P DE target genes GO and KEGG regulatory network, membrane raft (GO:0045121) process and focal adhesion (bta04510) pathways were in the central location. ITGB3 gene connected many pathways and formed the network, revealed the essential role of this hub genes (Fig.10 D-F). All the enrichment results demonstrated that the male sterility of cattle-yak may associated with lack of the regulation of such processes.

Table 2 The list of differentially expressed miRNA and their target genes of H vs P.

MiRNA id	Regulated	Target genes
bta-miR-122	up	HFE, CD151, SEMA4D, ADAMTS14,
bta-miR-146a	up	YTHDF2
bta-miR-184	up	EIF2S2, C7H5orf15, STOML2, FGFR1OP,
bta-miR-34c	up	ESPL1, KDM6A, THBS2, BCORL1, ZNF568, ALKAL2,
bta-miR-7	up	MYRFL, FANCA, INSL3, USP9X, SHF
bta-miR-93	up	SUFU, LOC789812, LOC789787, RALGAPA1, SEPHS1, NETO2, TMEM187, USP32, STOX2, RPAP2
bta-miR-455-3p	down	SMAD2, CAMKV, LDAH, GREB1, ANKS6, XRN1, CLEC7A, PIK3R1, PHF7, LOC787074, SPHK2, LINS1, MKRN3, IFIT3, RPAP2, TRPM2, ZNF217

Table 3 The list of differentially expressed miRNA and their target genes of M vs P.

MiRNA id	Regulated	Target genes
bta-miR-31	up	SUPT4H1, TWISTNB, ITGB1BP2, MICAL2, USP34, NR5A2, LOC112445404, DIS3
bta-miR-7	up	MYRFL, FANCA, INSL3, USP9X, SHF
bta-miR-193b	down	BTK, CALU, SLC39A5, GPR61, SLC25A19, USP21, GJC2, POLR3GL, RBFOX3, SERPINB9, ANKRD54, WFIKKN2, ACBD6
bta-miR-196b	down	POC1A, LCAT, IMPDH1
bta-miR-34a	down	HK1, GPAA1, SLA, IRF3, EIF2B4, SERPINB8, GSTT2, TMLHE, SLC13A4, MGC133764, MRTO4, CDK18, FBH1, EXTL1, INPP5K, RIMS2, RGCC, LYPD5, ZNF771, CDC73, GSC, PRSS12, SS18L2, LRRC8E, TNR, BAHD1, ITGB3, NRDE2, NTN5, CYP2D14, ADRB1, SZT2, CCNJL, SIGLEC1, SPEN, LOC530348, RTP3, CRYBG1, TMEM229B, HSPA14, CNNM2, FAM120B, CABIN1, LOC525426, MAP4, C18H19orf84, ATRNL1, STOX2, CHADL, RIMS4

3.6 qPCR validation

To confirm the relative expression levels of DE miRNAs and their target genes were consistent with the RNA-seq results, we selected the DE miRNA bta-miR-449a, the target genes FANCA, MYRFL and SHF of bta-miR-7 to perform the qPCR validation. We found that the miR-449a relative expression of cattle-yak was significantly lower than cattle and yak. And the target genes expression levels of FANCA, MYRFL and SHF were significantly up-regulated in cattle-yak, which is consistent with the lower expression of cattle-yak and negative regulation of bta-miR-7(Fig 11).

As important post-transcriptional non-protein-coding regulators, miRNAs engaged in many cellular and biological processes such as proliferation, apoptosis, differentiation and tumorigenesis.

MiRNA most mediate negative post-transcriptional regulation of their targets by base pairing to sequence motifs in the 3′ UTR of mRNAs as epigenetic modifiers[36–38]. It is elucidated that individual miRNA is able to control the expression of more than one target mRNAs and that each mRNA may be regulated by multiple miRNAs[39]. Over the past few decades, the studies that focus on the regulation of reproductive processes by miRNAs is gradually increasing. MiRNA-34/449 family members has been verified that play crucial roles for successful spermatogenesis, regulating spermatozoa maturation and functionality, the dysregulation of ciliogenesis in the efferent ductules is significantly impaired, leading to sperm aggregation and agglutination as well as to defective reabsorption of the seminiferous tubular fluids. Then the efferent ductules was obstructed and tubular fluids were accumulated, resulting to high hydrostatic pressure into the testis and then leads to testicular dysfunction and spermatogenic failure[40]. Besides, a previous study used the small RNA sequencing technology to reveal the differentially expressed miRNAs in High- and Low-motile sperm populations of cryopreserved semen and found that the DE miRNAs miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p may related to reproductive processes[41]. Thus, it can be seen that miRNAs could play great roles in regulating reproductive traits. Although there were systemically studies that related to the problem of male sterility of cattle-yak, the comprehensive exploration of compare the miRNAs profiles with cattle-yak and their parents’ generations were rarely can be seen.

In this study, we compared the DE miRNAs and DEGs among cattle, yak and cattle-yak groups. In H vs P groups, we found total 47 up-regulated and 34 down-regulated miRNAs and there were 7 known miRNAs (6 up-regulated and 1 down-regulated) can be mapped to their targets. The target genes ESPL1, KDM6A and BCORL1 may related to reproductive processes. In many previous studies, ESPL1(extra spindle pole bodies like 1) has been found that engaged in cellular mitosis and cell cycle process[42–44].KDM6A (lysine demethylase 6A) belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases, which is an X-linked protein that contains a catalytic Jumonji C (JmjC) domain which facilitates the removal of the methyl group on di- and trimethylated H3K27 (H3K27me2/3), it can not only manifested demethylase activity, but also regulated gene expression and enhancer activation independently of its demethylase activity. Many studies had demonstrated that the function of KDM6A in differentiation, development, regeneration and tumor suppressed [45–47].BCORL1 was also be proved that can be a contributor to spermatogenesis[48]. Furthermore, we merged the common differentially expressed miRNAs between the two comparison groups and found that the known miRNA bta-miR-7 was down-regulated in cattle-yak. With the analysis of target genes, we found that the FANCA gene is associated with the processes of DNA damage repair and the variation of FANCA would induce premature ovarian insufficiency and further cause female fertility[49, 50]. In this study, we quantified the expression levels of FANCA gene in cattle, yak and cattle-yak and detected higher relative expression level in cattle-yak. We concluded that the FANCA overexpression would increase the DNA stability excessively and interfere the processes of spermatocytes meiosis. Besides, the overexpression of SHF and MYRFL may also influence the spermatogenesis.

Furthermore, we performed GO, KEGG and GSEA analysis to annotate the function of the DE miRNAs target genes and DEGs. In the GO and KGEE enrichment annotation, we found that the up-regulated miRNA target genes in cattle and yak were most engaged in negative regulation of ubiquitin-dependent protein catabolic process, positive regulation of cell migration, positive regulation of mitochondrial DNA replication, positive regulation of DNA demethylation, protein deubiquitination processes and cell cycle (bta04110), PI3K − Akt signaling pathway(bta04151), RNA polymerase (bta03020), Focal adhesion (bta04510) and relaxin signaling pathway (bta04926).In our previous study, we have described the function of lncRNA target genes preliminarily[35]. In this work, we further used gene set enrichment analysis (GSEA) to expatiate the function of DE gene sets. We performed GSE GO and GSE KEGG to annotate the function of DEGs respectively and found that the DE gene sets were enriched in ATP binding, developmental process in reproduction, DNA binding, transporter activity, extracellular region processes and MAPK signaling pathway, pathways in cancer, cytokine receptor interaction and focal adhesion pathways. Studies had proved that both noncatalytic ATP binding and stable ADP binding can mediate chemo-mechanical transduction in axonemal dynein[51].ATP levels were highly associated with the levels of GSK3α sperm hexokinase activity[52]. As an efflux pump of ABCG subfamily of the ATP-binding cassette (ABC) transporters, ATP-Binding Cassette Transporter G2(ABCG2) was also detected in the blood-testis barrier and epididymal, which be proved that play a great role in epididymal sperm maturation[53–55].Mitogen-activated protein kinases (MAPKs) are important signal transducing enzymes that are involved in many facets of cellular regulation such as gene expression, cell proliferation, cell differentiation, programmed cell death and cell motility[56].MAPK signaling pathway can regulate dynamics of tight junctions and adherens junctions, proliferation and meiosis of germ cells, proliferation and lactate production of Sertoli cells[57, 58].Focal adhesion kinase is a widely expressed protein-tyrosine that play an important role in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix[59]. Focal adhesion pathways of focal adhesion kinase mediated has been implicated in a diverse array of cellular processes, such as cell migration, growth factor signaling, cell cycle progression and cell survival[60]. Such the processes and pathways in cattle and yak up-regulated target genes and gene sets may positively engaged in the sperm cells proliferation and seminal tubules differentiation.

Spermatogenesis is a complex and finely regulating process which is extremely important for racial reproduction. Spermatogenesis process contains spermatocytogenesis (mitosis), meiosis and spermiogenesis[61]. With a cycle of several mitotic divisions, spermatogonia and primary spermatocytes were produced and stem cells were renewed. In the stage of meiosis, the genetic material was duplicated and exchanged and then four haploid round spermatids were produced. Lastly, the haploid spermatids were differentiated and mature spermatozoa released into the lumin of seminiferous tubules. However, male hybrid of cattle and yak cannot finish the above processes completely. Histological examination of seminiferous tubules revealed that gonocytes and spermatocytes were established normally, however, spermatogenesis was arrested at the meiosis phase began at 10 months after birth in the hybrids[62].Previous studies also demonstrated that although cattle-yak have the same number of chromosomes (2n = 60) with that of cattle and yak, most of primary spermatocytes of cattle-yak performed morphologically abnormal and have no XY bivalents, which hindered the subsequently processes of meiosis and spermiogenesis[63]. The genes that regulating meiosis such as MEI1, SYCP3 and DAZL, has been detected significantly down-regulated in adult cattle-yak testes compared to those in yak testes[24, 64, 65].Besides, the target gene USP9X identified in this study is essential for proper spermatogenesis, USP9X-null spermatogenic cells underwent apoptotic cell death at the early spermatocyte stage, and then, caused subsequent aberrant spermiogenesis, which resulted in a complete infertility of USP9X conditional knockout male mice[66]. The comprehensive regulation of such miRNAs target genes and DE genes would disturb the spermatocytes meiosis process and further influence the reproductive traits of cattle-yak.

In this study, we used high-throughput sequencing technology to explore miRNA expression profiles in testicular tissues of cattle, yaks and cattle-yak, and conducted GO and KEGG enrichment analysis for their target genes. At the same time, we performed GSEA to annotate the gene sets of differentially expressed mRNAs among our nine samples. Finally, 47 up-regulated miRNAs and 34 down-regulated miRNAs were identified in cattle samples between the comparison of cattle and cattle-yak;16 up-regulated and 21 down-regulated miRNAs were identified in yak samples between the comparison of yak and cattle-yak. In our previous description, we have demonstrated that the pathways of the target genes and the function of DE gene sets are most associated with the reproductive regulation. Subsequently, we merged the same differentially expressed miRNAs between H vs P and M vs P. As a result, the known miRNA miR-7 was commonly down-regulated in cattle-yak in the two comparison groups. This suggests that miR-7 may play an extremely important role in reproductive processes. The target genes predicted in this study such as MYRFL, FANCA, INSL3 and USP9X of miR-7 may regulate the reproductive traits synergistically. Our research will be beneficial to know about the molecular mechanism of cattle-yak male sterility and provide better methods to resolve it. We will carry out more deep going researches concentrated on the DE genes and the target genes.

Ethical Statements

We disclaimed that the number of animals in this study was minimum and special attention was paid to minimize animal suffering. The experiment designs and the samples collection involved in this study correspond to the requirements of ARRIVE guidelines 2.0[67].

Animals and testis sample collection

Three male cattle (H1, H2, and H3), three male cattle-yaks (P1, P2, and P3) and three male yaks (M1, M2, and M3) of consistent diet levels, with the same growth conditions, aged at 18 months were randomly selected on a pasture of Hongyuan country, Sichuan province of China. They were then divided into three groups (H, P and M). Testicular tissue samples were collected and stored at −80 ◦C until total RNA was extracted when these animals were slaughtered.

Histological analysis of testicular tissues

Testicular tissues collected from nine samples were immersed in 4 % phosphate-buffered formalin overnight. Then the fixed paraffin-embedded tissues were sectioned with 4μm thickness. The sections further been stained with hematoxylin and eosin (HE) and toasted in the oven set to 70℃.

RNA extraction, library preparation and sequencing

Total RNA was extracted from nine testicular tissue samples with Total RNA Extractor(Sangon Biotech, Shanghai, China).Then Qubit2.0（Life Technologies, Shanghai, China）and agarose gel were used to detect the concentration and integrity of total RNA, respectively. Total RNA was quantified accurately and then the 3′-adapter and 5′-adapter were ligated to each end of the miRNA, then RT reaction was used to create single stranded cDNA. The cDNA is amplified through PCR and 12%-page gel was used to detect the integrity of the library. The 140-150 bp libraries sequencing were carried out on the illumina platform (Illumina Hiseq X Ten).

Quality control, reads mapping and sequence analysis

FastQC was used to quality checks on raw data of nine samples. Cutadapt and trimmomatic[68] were then used to remove the adaptors and low-quality reads, respectively. Blastn was used to aligned to Rfam[69] database and then the reads that mapped to the bovine rRNA, sRNA, snRNA, snoRNA were discarded. Bowtie[70] was used to mapped to bovine reference genome, exon and intron sequence, the reads that can be mapped to the bovine genome and the intron region were retained. Novel miRNA prediction and known miRNA quantification were separately ran with the miRDeep2 module and quantifier module of the miRDeep2[71] software.

Differential expression analysis

The R package edgeR[72] was utilized to compare the expression level of miRNAs between H_vs_P and M_vs_P. Specifically, the lowly expressed miRNAs were filtered with CPM normalization method, the DE miRNAs among cattle, yak and cattle-yak were screened with P-value < 0.05, |fold change| >2. We also carried on the venn analysis to merge the common DE miRNAs between H_vs_P and M_vs_P for further analysis.

Target prediction and functional analysis of DE miRNAs

Miranda was used to predict the target genes of DE miRNAs of H_vs_P and M_vs_P. ClusterProfiler[73] was used for enrichment analysis of biological processes, cellular component and molecular function in Gene Ontology and pathway in KEGG. At the same time, we concentrated on the target genes of common miRNAs among cattle, yak and cattle-yak to carry on functional enrichment analysis

GSEA of DE mRNA

In our previous study, we have already preliminarily explored the function of the novel lncRNA target genes and differentially expressed genes (DEGs) with GO and KEGG enrichment analysis[35]. In this study, we further utilized the gene set enrichment analysis (GSEA) to understand the function of the DE gene sets more accurately and deeply with R packages msigdbr and clusterProfiler[74].

Net work analysis of DE miRNA target genes

In order to understand the interactions among function processes and genes with pathways intuitively, igraph was used to draw the net plots and describe the correlations among pathways. Specially, the GO to GO, gene to pathways and pathway to pathway plots were diagramed, respectively.

qPCR validation and Statistical analysis

To verify the accuracy of sequencing results, miRNA and several target genes were randomly selected for qPCR validation. The identified DE miRNAs miR-449a and the target genes FANCA, MYRFL, SHF of miR-7 were selected to the further molecular experiment. The 2 ^-ΔΔCTmethod was used to quantified the relative expression of the miRNAs and the target genes. Student’s t-test with Graph Pad Prism 6 software was used to compare the differences among cattle, yak, and cattle-yak samples. *p-value < 0.05 was considered statistically significant and **p-value < 0.01 was considered as extremely statistically significant.

miRNA: micro RNA

mRNA: message RNA

GO: Gene Ontology

KEGG: Kyoto Encyclopedia of Genes and Genome

GSEA: Gene Set Enrichment Analysis

DEGs: Differentially Expressed Genes

RPM: Reads Per Million.

CPM : Counts Per Million

H: Cattle

M: Yak

P: Cattle-yak

3′ UTR: 3’ Untranslated Region

qPCR: quantitative polymerase chain reaction

Ethics approval and consent to participate

The study design was approved and all methods were performed in accordance with

guidelines of the Institutional Animal Care and Use Committee at the College of Animal

Science and Technology, Sichuan Agricultural University (permit number: DKY2020050). The experiment designs and the samples collection involved in this study correspond to the requirements of ARRIVE guidelines 2.0[67]. The minimal number of animals was used and special attention was paid to minimize animal suffering.

Consent for publication

Not applicable.

Availability of data and materials

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA006824) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa [75, 76].

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the National Key R&D Program of China (2021YFD1200403), Sichuan innovation team of national modern agricultural industry technology system (SCCXTD-2022-13), and the Key R&D Program of Sichuan Province (2021YFYZ0007).

Authors' contributions

SKZ and WES performed the data analysis and drafted the manuscript. SKZ, XBJ and YCL collected the testicular samples involved in this study. YCL contributed to laboratory experiments. SJL and SYC revised the manuscript. XBJ and JW designed the study and also revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Authors' information

Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, China

Shaokang Zhao, Wenqiang Sun, shiyi Chen, Yuchao Li, Jie Wang, Songjia Lai & Xianbo Jia

Corresponding author

Correspondence to Xianbo Jia

Supplementary Information

Can be seen as supplementary materials.

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No competing interests reported.

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The exploration of miRNAs and mRNAs profiles revealed the molecular mechanisms of cattle-yak male infertility

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