Analysis of RNA Population in Goat Spermatozoa.

Abstract

Whole transcriptome studies in humans, rodents, and livestock species such as cattle, pigs, and horses have shown the presence of both coding and non-coding RNAs in spermatozoa. However, a detailed analysis of the spermatozoal RNA (spRNA) population in goats and their relevance to sperm functions, such as motility and capacitation, is lacking in the literature. This study investigated the RNA population in goat spermatozoa by RLM-RACE, long-read NanoporeTM sequencing, and short-read IlluminaTM sequencing. The effects of heparin- and arginine-induced sperm capacitation and motility enhancement on spRNAs were also investigated. Results showed that motile spermatozoa can be best recovered (89.20±1.15%) by the Swim-up method and could maintain sperm motility (82.33±1.53%), viability (88.10±5.03%), and plasma membrane integrity (71.33±4.51%) in sperm TALP (Sp-TL) medium for at least 1 h. Treatment of sperm samples with 0.5% (v:v) Triton-X and 0.1% (v:v) sodium dodecyl sulfate completely removed the contamination of somatic cells, as revealed by negative expression of PTPRC and positive expression of PRM1 and PRM2 upon reverse-transcriptase polymerase chain reaction (RT-PCR). The goat spermatozoa were found to contain 20.03±0.47 fg of RNA per cell. The addition of dithiothreitol (DTT) as a reducing agent (20 mM) to the monophasic solution of GITC and phenol increased the yield of large-sized RNA (3.89±0.46 ng/million cells) suitable for long-read RNA sequencing (RNA-seq) of poly(A) transcripts. NanoporeTM sequencing resulted in reads ranging from 500bp to 2Kb in length that could be mapped to 123 transcripts by de novo assembly. On the other hand, full-length cDNA cloning by RLM-RACE identified 31 transcripts. The full-length transcripts of five fertility-related genes (AR, CATSPER3, DAZL, HSP90AA1, SPACA1, PRM3) were found in the goat spermatozoa. The short-read RNA-seq IlluminaTM discovered 16,604 transcripts that included mRNA (92%), lncRNA (4%), rRNA (2%), circRNA (1%), miRNA (1%), etc. A total of 357 mRNAs had FPKM of more than five. The goat spRNA population was also predicted to have 127 circRNAs, 655 lncRNAs, and 160 imprinted genes. The coding mRNAs were involved in different biological pathways, viz. Wnt signaling, cAMP-signaling, AMPK signaling, and MAPK signaling pathways. Results further showed that heparin-induced capacitation upregulated 1,251 transcripts and included PRND, LLCFC1, and SPESP1 genes that are known to participate in signaling pathways of sperm capacitation and acrosome reaction. Similarly, arginine-induced sperm capacitation and motility enhancement were also associated with the upregulation of 1,309 genes. The differentially expressed genes (DEGs) in both the heparin- and the arginine-treated group had upregulated genes involved in cAMP/PKA signaling, PI3-Akt signaling, MAPK signaling, calcium signaling, and oxidative stress pathways. DCFDA staining confirmed the increased generation of reactive oxygen species (ROS) in goat spermatozoa treated with heparin or arginine. In conclusion, this study documents the suite of RNA populations in goat spermatozoa and their relevance to heparin- and arginine-induced sperm capacitation. A molecular mechanism of heparin- and arginine-mediated sperm capacitation is proposed. This is the first report on RNA-seq of goat spermatozoa and their relevance to sperm function. Future studies should determine the existence of de novo transcriptional activities in goat spermatozoa during in vitro capacitation.