Solexa Profiling Identifies Differentially Expressed MiRNAs Between Sexually Immature and Mature Equine Testis

1 Human and Animal Health Vol.61: e18160122, 2018 http://dx.doi.org/10.1590/1678-4324-2018160122 ISSN 1678-4324 Online Edition BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Solexa Profiling Identifies Differentially Expressed MiRNAs Between Sexually Immature and Mature Equine Testis Liangjun He1, Shiwei Wang2, Haifeng Deng3, Hong Dong1, Jingbo Chen4*. Shihezi University – College of Animal Science, Shihezi, Xinjiang, China; 2Tarim University – College of Animal Science, Aral, Xinjiang, China; 3Zhaosu Horse Farm, Yili, Xinjiang, China; 4Xinjiang Academy of Animal Science – Institute of Animal Science, Urumqi, Xinjiang, China . 1 ABSTRACT MicroRNAs (miRNAs) are a class of short non-coding RNAs identified as potent regulators of gene expression. Previous studies have suggested that miRNAs are involved in mammalian spermatogenesis. Stallion fertility is an important trait for the horse breeding industry, but stallion fertility traits are largely ignored in the industry. In this study, we generated expression profiles of miRNAs in foal (immature) and stallion (mature) testes using Solexa sequencing. We identified 438 known and homologous equine miRNAs and 199 novel miRNAs which were distributed among all the chromosomes. The two developmental stages showed significant differences in miRNA expression patterns. Our result expands the horse miRNA database and provided additional information on the stallion fertility and possible spermatogenesis regulation through specific miRNAs. Key words: horse; miRNAs; solexa; testis * Author for correspondence: Braz. Arch. Biol. Technol. v.61: e18160122 2018 2 Chen, J. et al INTRODUCTION MicroRNAs (miRNAs) are short, non-coding, endogenous RNAs, 19 to 25 nucleotides (nts) in length, which have been identified as potent regulators of gene expression through post-transcriptional gene silencing. The first miRNA identified, lin-4, was found in the nematode Caenorhabditis elegans by standard positional cloning of genetic loci and is involved in developmental timing [1]. Biosynthesis of miRNAs begins with the transcription of the primary miRNA (pri-miRNA) by RNA polymerase II. The primiRNA is recognized and cleaved by a microprocessor complex, Drosha and DGCR8, to produce a stem-loop RNA (pre-miRNA), which is exported from the nucleus by exportin 5 into the cytoplasm and is then cleaved by Dicer to yield a double-stranded RNA. Subsequently, the double-stranded RNA is separated into single strands, and generally one of the two strands is incorporated into the RISC complex, while the other strand is degraded [2]. Most commonly, the RISC-incorporated miRNA binds to the 3'UTR of the target mRNA by base-pairing and consequently induces either translational repression or mRNA degradation [3]. Male fertility requires that there are large numbers of normal spermatozoa in the testis, formed though a complex process known as spermatogenesis. Spermatogenesis is a precisely synchronized process, which involves mitotic cell division and propagation of spermatogonial stem cells (SSCs), meiotic division, and subsequent processing in the seminiferous tubules. The division of type A spermatogonia provides both self-renewal of SSCs and type B spermatogonia, which differentiate and divide mitotically into primary spermatocytes. During meiosis, primary spermatocytes divide into two secondary spermatocytes and then produce four haploid round spermatids, which contain half the original number of chromosomes. Finally, haploid cells undergo a morphologic transformation known as spermiogenesis to develop into mature spermatozoa. Many studies have suggested that miRNAs are involved in spermatogenesis. The deletion of the Dicer gene (encoding an enzyme required for miRNA biogenesis) in mouse primordial germ cell results in retarded spermatogenesis, which demonstrates that this miRNA is essential for primordial germ cell and spermatogonia proliferation [4]. miRNA are also important for the late stages of spermatogenesis. The knockout of Dicer1 in mouse germ cells causes decreasing germ cell number in the seminiferous tubules, impaired transition from round to elongated spermatids and abnormal sperm motility [5]. Immature and mature testes have different miRNA expression profiles, and many miRNAs are stage-specifically expressed in spermatogenesis. Yan et al. identified sox5 and sox6 as presumed targets of miR-181c and rsbn1 as putative target of miR-355, miR-181c and miR-181b [6]. The first study on equine miRNA identified 407 novel horse miRNA genes corresponding to 354 mature miRNAs, using a comparative genomics approach [7]. Illumina Next Generation Sequencing technology was used to identify 292 known miRNAs and 329 novel miRNAs in horse skeletal muscle, colon and liver tissues [8]. In order to discover sperm-based biomarkers for stallion fertility, sperm and testis transcriptomes were compared using microarray and RNA-seq [9]. In that study, the researchers found 6761 transcripts in sperm and 11,112 in testis, including 82 sperm miRNAs. Despite these efforts, complete miRNA expression profiles and functional annotation of miRNAs during spermatogenesis have not been characterized in horse. A horse aged one year or younger is called a foal. At approximately 1.5 yr of age, stallions reach puberty. Spermatogenesis is completed at 2 to 3 years of age. At four years old, stallions are mature in terms of testicular weight, daily sperm production and Braz. Arch. Biol. Technol. v.61: e18160122 2018 3 Solexa Profiling Horse Testis miRNAs Sertoli cell number [10]. Sub-fertility in stallions increases veterinary fees and management costs and ultimately diminishes the genetic contribution from prized stud horses[11]. Therefore, understanding the differential miRNA expression between mature and immature equine testes may lead to a new direction in the search for biomarkers for stallion fertility and treatments for stallion infertility. In this study, we used Solexa deep sequencing technology to characterize and compare miRNA expression profiles between sexually mature and immature horse testis to discover miRNA biomarkers for stallion fertility. As a result, we identified 438 known and homologous equine miRNAs and 199 novel miRNAs. These results show that the two testicular developmental stages have significantly different miRNA expression patterns that can be used as biomarkers of testicular maturity. MATERIALS AND METHODS Tissue Collection In order to ensure the maturation of the testes, three Kazakh stallions (5–10 years old), who lived with a herd of mares (n>20) in pasture during the breeding season in previous year and the pregnancy rate of each mare herd was higher than 70%, were selected. Samples from these stallions were used as mature testes. Additional histological examination of all mature testes all showed normal spermatogenesis. Additionally, three normal Kazakh foals (immature, 5–8 months old) were castrat (...truncated)