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Chicken Sperm Transcriptome Profiling by Microarray Analysis Genome Chicken sperm transcriptome profiling by microarray analysis Journal: Genome Manuscript ID gen-2015-0106.R2 Manuscript Type: Article Date Submitted by the Author: 29-Nov-2015 Complete List of Authors: Singh, R. P.; Salim Ali Centre for Ornithology and natural History, Avian Physiology and genetics Shafeeque, C M; Salim Ali Centre for Ornithology and Natural History, ; rajiv gandhiDraft centre for biotechnology, HMG Sharma, S; Central Avian Research Institute, Physiology and Reproduction Singh, Renu; Indian Veterinary Research Institute, Mohan, J; Central Avian Research Institute, Physiology and Reproduction Sastry, K; Central Avian Research Institute, Physiology and Reproduction Saxena, V; Central Avian Research Institute, Azeez, P; Salim Ali Centre for Ornithology and natural History, Avian Physiology and genetics Keyword: fertility biomarker, sperm RNA, embryonic development, egg, fertilization https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 158 Genome Chicken sperm transcriptome profiling by microarray analysis R.P. Singh 1,*, C.M. Shafeeque 1, S.K. Sharma 2, R. Singh 3, J. Mohan 2, K.V.H.Sastry 2, V. K. Saxena 2, P. A. Azeez 1 1Avian Physiology and Genetics Division, Sálim Ali Centre for Ornithology and Natural History, Anaikatty-641108, Coimbatore, India 2Central Avian Research Institute, Izatnagar, 243122, India. 3Indian Veterinary Research Institute, Izatnagar, 243122, India. Draft Running title: RNA in chicken sperm *Corresponding author current address Smithsonian Conservation Biology Institute, Front Royal, VA 22630 E-mail address: [email protected] Phone- +1-540-305-5911 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 158 Abstract It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from ten fertile males and used for RNA preparation.Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 x 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 17542 predominantlyDraft nuclear-encoded transcripts in chicken sperm. The majority (87.3%) of the sperm transcripts were shared with the testes, while surprisingly, 12.7% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility. Key words: fertility biomarker, sperm RNA, embryonic development, egg, fertilization https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 158 Genome Introduction Historically, it was believed that spermatozoa are just a vehicle that delivers the male genome to the oocyte. However, in the last decade research has shown that spermatozoa delivers more to the oocyte than just the paternal haploid genome. Upon fertilization, the spermatozoon provides a complete, highly structured, and epigenetically marked genome that, together with a defined complement of RNAs and proteins, serves a variety of divergent functions (Krawetz, 2005). A recent study reported an absence of fertility-related sperm RNA elements in men with idiopathic infertility, but whose sperm characteristics were normal (Jodar et al. 2015). Zhang et al. (2006) reported that infertile men possess a higher proportion of spermatozoa with an increased histone to protamine (PRM) ratioDraft than fertile controls. Similarly, human sperm devoid of phospholipase C zeta 1 (PLCZ1) fail to induce Ca 2+ release and are unable to initiate the first step of embryo development (Yoon et al. 2008). Studies have reported that sperm antisense RNAs can epigenetically regulate early embryonic development and have a structural role in maintaining histone-bound sperm chromosomal regions (Lalancette et al. 2008; Krawetz et al. 2011; Liu et al. 2012). Besides their role in fertilization, Gapp et al. (2014) provided evidence for the idea that sperm RNA-dependent processes contribute to the transmission of acquired traits in mammals by experimentally proving that sperm RNAs are responsible for transgenerational inheritance of the effects of early trauma in mice. The transcriptional profiling of sperm RNAs has been done for humans, mice and other domesticated animals such as bovine, swine and stallion (Goodrich et al. 2007; Gilbert et al. 2007; Yang et al. 2009; Das et al. 2013; Kawano et al. 2012; Card et al. 2013), and those results indicate a combination of coding, non-coding and miRNA molecules present in sperm. Gilbert et al. (2007) compared between bovine sperm and spermatid transcriptome and reported that sperm https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 158 harbors a complex mixture of messengers implicated in a wide array of cell functions and representing a large subset of transcripts found in spermatids. Another study reported many full- length transcripts in bovine sperm, the presence of which have already been reported in sperm of other species (Card et al. 2013), indicating that some candidate sperm transcripts might be conserved across species. Das et al. (2013) analysed the transcriptome of stallion sperm vs. testes by microarray and RNA-sequencing, and those results suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs. These results confirmed that sperm not only transfer the haploid male genome to the oocyte, but also deliver specific paternal messenger and non-coding RNAs to the egg at fertilization. However, the overall functional significance of these sperm RNAsDraft in fertilization, early embryonic development or in any other function is yet to be understood and elucidated in most species. As a first step to examining the functional significance of sperm RNAs in any species, it is essential to characterise the species-specific sperm enriched RNA population, which will help in determining the molecular functions of sperm RNAs in silico based on biological pathway analysis. Therefore, this study was conceived to explore the chicken sperm RNA profiling by comparison of RNA transcripts between spermatozoa and testes using a microarray technique. Materials and methods All the procedures used in this study were reviewed and approved by the Central Avian Research Institute Animal ethics committee and were carried out in accordance with the revised framework of animals (Scientific Procedures) Act of 2002 of Government of India on animal welfare. https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 158 Genome Semen and testes sample preparation Semen from ten fertile male broilers (IC3) of the same hatch (about 40 weeks of age) was collected by the abdominal massage method (Quinn and Burrows 1936), and evaluated for motility as described by Wheeler and Andrews (1943) and fertility by natural mating. The first few ejaculates from each male were discarded in order to obtain good quality semen. Semen samples that showed a motility score above 3 were used for further processing. The concentration of spermatozoa was determined spectrophotometrically at 550nm as described by Brillard and McDaniel (1985). To eliminate variation between individuals, an equal volume of semen from all males was pooled and considered as a single sample, and subjected for density gradient centrifugation. After semen collection,Draft all ten males were euthanized, testes were removed aseptically and washed with RNase free chilled phosphate buffer saline. Approximately 1 mg of testicular tissue from each male was collected, pooled and mixed with 1 mL of RNAlater and stored at 4 0C for further use. Two biological replicates from one pooled sample of each sperm and testes RNA was used for microarray study. Somatic cell removal by density gradient centrifugation Pooled semen was subjected to density gradient centrifugation (DGC) using density gradient mediums, PureSperm (Nidacon International, Mölndal, Sweden), for the removal of somatic cells and immature diploid spermatocytes. In brief, 400 µL neat semen was mixed well with 1000 µL of 1X PBS and centrifuged at 1000 g for 5 min at room temperature (RT). The resultant pellet was then again dissolved with 1000 µL 1X PBS, and sperm concentration was measured and adjusted. An aliquot of 1000 µL (500-800 x 10 6 sperm) from this resultant semen solution was layered over 3 mL pre-warmed (37 °C) 40% PureSperm and centrifuged at 280 g for 30 min https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 158 at RT. The bottom layer was carefully aspirated using a Pasteur pipette without disturbing the upper layer, transferred into a 2 mL centrifuge tube and washed twice with 1X PBS by centrifuging at 1000 g for 5 min at room temperature. Finally, the sperm pellet was suspended in 1 mL 1X PBS, and
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