Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (20): 4297-4312.doi: 10.3864/j.issn.0578-1752.2020.20.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Molecular Characterization of Tibetan Sheep BOLL and Its Expression Regulation and Functional Analysis in Testis

LI TaoTao1(),WANG Xia1,MA YouJi1,2(),YIN DeEn1,ZHANG Yong3,ZHAO XingXu3   

  1. 1College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070
    2Sheep Breeding Biotechnology Engineering Laboratory of Gansu Province, Minqin 733300, Gansu
    3College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070
  • Received:2020-06-01 Accepted:2020-09-03 Online:2020-10-16 Published:2020-10-26
  • Contact: YouJi MA E-mail:ttli2018@163.com;yjma@gsau.edu.cn

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Abstract:

【Objective】 As a RNA-binding protein, BOLL exerts integral roles during spermatogenesis through interactions with other molecules. The present study was aimed to analyze the sequence characteristics of Tibetan sheep BOLL and its expression and distribution patterns in testis which, in turn, explored the regulation and potential biological function of BOLL expression, hoping to offer much needed perspective and molecular insight for deciphering the mechanism of BOLL during sheep spermatogenesis in the future. 【Method】Eight healthy male Tibetan sheep from each of three key developmental stages, including 3 months old (sexually immature), 1 year old (sexually mature) and 3 years old (adult), were selected. The full-length coding sequence (CDS) region of Tibetan sheep BOLL was cloned by real time PCR (RT-PCR) using total RNA from the right testis samples; the sequence and structural signatures of BOLL and its interacting proteins were analyzed via relevant bioinformatics software; the expression and immunolocalization characteristics of BOLL in testicular tissues at three developmental stages were assessed with quantitative RT-PCR (qRT-PCR), Western blot, and immunofluorescence staining; the competing endogenous RNA (ceRNA) regulatory network and GO functional annotation for ovine BOLL were investigated with the aid of related databases, based on previous data of an integrative analysis of transcriptional profiles from Tibetan sheep testicular tissues by our group, and their expression patterns and targeting relationships were verified by qRT-PCR and dual luciferase reporter assay, respectively. 【Result】 The full CDS region of Tibetan sheep BOLL was 888 bp in length, capable of encoding 295 amino acids which contained a RRM domain of 81 amino acids (near the N-terminal) and a DAZ repeat motif of 25 amino acids. Ovine BOLL exhibited high sequence homology and evolutionary conservation with other mammals, especially for goat, cattle, and yak. There was the potential interaction between sheep BOLL protein and 10 proteins associated with the development of male germ cells. With increasing age, mRNA expression of BOLL was consistently up-regulated in Tibetan sheep testes, while protein expression of which was up-regulated followed by down-regulation. BOLL protein predominantly existed in spermatids from post-pubertal (1 year old and 3 years old) testes, and small amounts were present in spermatocytes, as well as spermatogonia in testes throughout development stages. The qRT-PCR results showed that compared with 3 months old, the expression levels of microRNAs (miRNAs) oar-miR-127-5p、oar-miR-382-5p and oar-miR-760-3p exhibited an extremely significant reduction (P<0.01) , while the expression levels for long noncoding RNAs (lncRNAs) LOC105602204, LOC105603195 and LOC105616228, as well as for circular RNAs (circRNAs) circ-ECT2L and circ-SPHKAP exhibited an extremely significant increase in 1 year old and 3 years old (P<0.01). oar-miR-127-5p and oar-miR-760-3p significantly decreased the luciferase activity of BOLL 3′-UTR wild-type, and oar-miR-760-3p significantly decreased the luciferase activities of wild-type Circ-ECT2L and wild-type LOC105616228 reporter genes. 【Conclusion】 This study was the first to report the molecular characteristics and expression patterns of BOLL gene and regulation of its expression in Tibetan sheep testis. BOLL was predominantly expressed in the post-meiotic round and elongating spermatids of Tibetan sheep, and the expression of which was directly targeted and negatively regulated by oar-miR-127-5p and oar-miR-760-3p; and was positively regulated by oar-miR-760-3p -mediated circRNA Circ-ECT2L and lncRNA LOC105616228. In turn, which might may interact with downstream signaling molecules to participate in the differentiation of ovine spermatids into mature spermatozoa.

Key words: BOLL, cloning, Tibetan sheep, testis, spermatogenesis

Table 1

Primer information used for qRT-PCR"

引物名称
Primer name		 上游引物
Forward primer (5′-3′)		 下游引物
Reverse primer (5′-3′)		 产物长度
Product length（bp）
BOLL GGCGCAAACATCAAATCAGAC GGGCACTCGTTGGGTTATTC 92
β-actin CTTCCAGCCTTCCTTCCTGG GCCAGGGCAGTGATCTCTTT 180
LOC105602204 ATATGACACGACGGGACAGC CACAACCCGGTGCGTATCTA 242
LOC105603195 GGGATTTGTCACTGGGCTCT TGTGTTCTTCCCATTCGCCT 258
LOC105616228 GTCTGGTCGGGAAATGCTGG GGGCTCTCGTAAAACCTCCC 156
Circ-ECT2L AGAGACTCATCTGGGGGTGC TCTGGTTCGCTTTTCGGCT 154
Circ-SPHKAP TTATTTCCGAATGCAGCCCC TCCTCCTTAGTGGTTTCCTTTT 182
oar-miR-760-3p CGGCTCTGGGTCTGTGGG Universal reverse* -
oar-miR-382-5p GAAGTTGTTCGTGGTGGATTC Universal reverse* -
oar-miR-127-5p CTGAAGCTCAGAGGGCTCTG Universal reverse* -
U6 GGAACGATACAGAGAAGATTAGC TGGAACGCTTCACGAATTTGCG -

Fig. 1

Cloning and sequence analysis of Tibetan sheep BOLL CDS region A: Structural diagram of the ovine BOLL (The sequence data is available from GenBank: accession No. NC_040253.1). B: PCR amplification derived from cDNA of Tibetan sheep BOLL gene. M, DL2000 marker. C: Open reading frame (ORF) analysis of Tibetan sheep BOLL cDNA amplification product. D: The nucleotide and deduced amino acid sequences for the cloned BOLL CDS region. ATG and TAA are the start codon and the stop codon, respectively"

Fig. 2

Comparative analysis of BOLL CDS sequence features among different mammals"

Fig. 3

Phylogenetic tree for amino acid sequences of BOLL protein based on Neighbor-joining algorithm The bootstrap values and branch lengths were showed above and below each branch, respectively. A closer phylogenetic relationship with Tibetan sheep BOLL is indicated by the asterisk"

Fig. 4

Spatial structure of Tibetan sheep BOLL protein A: Secondary structure; B: Tertiary structure"

Fig. 5

Network analysis of the proteins interacting with ovine BOLL protein The circles represent proteins, and the straight lines represent the interactions between different proteins. Circle size corresponds to the protein numbers of interacting with this protein, with larger circles indicating the greater the numbers of interacting-proteins. The line thickness denotes the confidence of interactions (confidence threshold 0.4), with thicker lines indicating higher confidence in the protein-protein interaction"

Fig. 6

Expression patterns of BOLL mRNA and protein in developmental Tibetan sheep testes A: Comparison of the changes in mRNA expression of BOLL acquired by RNA-seq and by qRT-PCR validation. All RNA-seq experiments consisted of four biological replicates; all qRT-PCR experiments consisted of eight biological replicates each consisting of three technical replications. The mRNA expression abundance of BOLL obtained from RNA-seq was expressed by the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) value. B: Western blot analysis for BOLL protein; C: The relative level of BOLL protein expression. 3M: 3-month-old; 1Y: 1-year-old; 3Y: 3-year-old. **: Extremely significant difference (P<0.01); ns: Non-significant difference (P>0.05)"

Fig. 7

Immunofluorescence staining of BOLL protein in Tibetan sheep testis A: Immunolocalization of BOLL protein; B: Negative controls. 3M: 3-month-old; 1Y: 1-year-old; 3Y: 3-year-old. DAPI (blue): Nuclear staining; FITC (green): Fluorescent staining; Merge: Colocalization"

Fig. 8

The functional annotation and potential ceRNA regulatory network for ovine BOLL"

Fig. 9

Temporal expression patterns of potential regulatory non-coding RNAs for BOLL gene and verification of the target relationship A: Comparison of the changes in expression of non-coding RNAs obtained by RNA-seq (four biological replicates) and by qRT-PCR validation (eight biological replicates each consisting of three technical replications). The expression abundance of miRNAs, circRNAs, and lncRNAs obtained from RNA-seq was measured by the TPM (Transcripts Per Kilobase of exon model per Million mapped reads), RPM (Reads of exon model per Million mapped reads), and FPKM (Fragments Per Kilobase of exon model per Million mapped fragments), respectively. B: A dual luciferase reporter assay was used to validate the targeting relationships between miRNAs and BOLL or their ceRNAs. 3M: 3-month-old; 1Y: 1-year-old; 3Y: 3-year-old. **: Extremely significant difference (P<0.01); *: Significant difference (P<0.05); ns: Non-significant difference (P>0.05)"

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