关键词: BOLL, 克隆, 藏绵羊, 睾丸, 精子发生

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