Identification and quantification of coding and long non‐coding RNAs in stallion spermatozoa separated by density

Abstract

Background It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. Objectives To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. Materials and methods Spermatozoa from five stallions were separated into more dense and less dense populations by density gradient centrifugation. Complementary DNA libraries were made from each of the ten total RNA samples after ribosomal RNA removal. Next‐generation sequencing characterized the RNA populations in more and less dense spermatozoa. Quantitative reverse transcription‐PCR was used to confirm differential expression of selected RNAs. Results Stallion spermatozoa contain 11 215 RNAs, with the most prevalent RNA being a 1492 base long non‐coding RNA. The levels of 159 RNAs were greater in more dense spermatozoa, while levels of seven other RNAs were greater in less dense spermatozoa. Quantitative reverse transcription‐PCR confirmed the threefold greater levels of solute carrier family 26 member 8 (SLC26A8) mRNA in less dense spermatozoa, and sixfold and threefold greater expression levels of the SCP2 sterol binding domain containing 1 (SCP2D1) and spermatogenesis‐associated protein 31D1 (SPATA31D1) mRNAs in more dense spermatozoa, respectively. Discussion and conclusion We identified 11 215 RNAs in stallion spermatozoa and 166 with differential expression between more dense and less dense fractions. Many prevalent RNAs were also found in bull, boar, and human spermatozoa. Many differentially expressed RNAs are known to be testis‐ or spermatozoa‐specific. Our results may lead to identification of an RNA population in spermatozoa that is optimal for establishing successful pregnancies.

Functions and mechanism of noncoding RNA in regulation and differentiation of male mammalian reproduction

“…Targeting interferon regulatory factor 1 (IRF1) an, therefore, downregulating CCND1, cyclindependent kinase 2 (CDK2), and cyclin-dependent kinase inhibitor 1A, miR-383 controls mitotic division in spermatogonia and early spermatocytes (p21). 29,30 Additionally, SSCs express miR-294 and miR-295, while premeiotic male germ cells only express miR-201 and miR-547. 31 This suggests that these miRNAs regulate NOTCH, Erk1/2, and PI3K…”

Long Noncoding RNAs: Recent Insights into Their Role in Male Infertility and Their Potential as Biomarkers and Therapeutic Targets

“…In large livestock, Wang et al [39] reported that lncRNAs are important in the regulation of bull sperm motility, and a total of 2517 lncRNAs were differentially expressed when compared between Holstein bulls with low or high sperm motility. In stallion sperm, 11,215 lncRNAs were identified, and 166 DELs were found to differ between groups of stallions living at greater and lower densities [40]. Because associations have been reported between many lncRNAs and reproductive disorders, it is very prudent to characterize and identify lncRNAs by sequencing and bioinformatic approaches as these may help us to determine the causes of male infertility [41].…”

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock

“…The latter may thus, suggest the importance of spRNAs in regulating protein translation during spermatogenesis ( Bettegowda and Wilkinson, 2010 ). Comparative transcriptomics of stallion sperm identified 149 DE miRNAs between dense and less-dense spermatozoa ( Ing et al, 2020 ) wherein BC O 1, GLRA4, OTOL1, PRM1, SCP2D1 , and SPATA31D1 were highly expressed in dense spermatozoa compared to those of less-dense spermatozoa. This study also found that expression of PRM1 transcripts was significantly higher in morphologically normal spermatozoa from sub-fertile stallions and in spermatozoa with abnormal morphology ( Paradowska-Dogan et al, 2014 ).…”

“…Many targets of spermatozoal lncRNAs have been predicted ( Gao et al, 2019 ) and were found to be enriched in apoptosis (e.g., PI3K-AKT, p53) ( Xiong et al, 2019 ) and capacitation-related pathways (e.g., Calcium, cAMP, and MAPK signaling) ( Gao et al, 2017 ). In stallion, comparative transcriptomics of dense and less-dense spermatozoa revealed 1,492 bp lncRNA as the most prevalent RNA ( Ing et al, 2020 ). Further, the expression of 159 RNAs was higher in dense spermatozoa than in less-dense spermatozoa.…”

“…Several studies also identified Calcium ion channels such as CatSper in the spRNA population, which is essential in regulating capacitation and sperm motility in livestock species as well as humans ( Jan et al, 2017 ; Li et al, 2018 ; Singh et al, 2019 ; Nicolas et al, 2020 ). Besides these common sperm transcripts across various livestock species, there were flagellar specific spRNAs and sperm motility proteins such as BSP ( de Souza et al, 2017 ; Pardede et al, 2020 ), AKAP ( Gilbert et al, 2007 ; Chatterjee et al, 2010 ; Kasimanickam and Kastelic, 2016 ; Ing et al, 2020 ), ODF ( Chen et al, 2015 ; Li et al, 2018 ; Pardede et al, 2020 ; Özbek et al, 2021 ), zinc finger nucleases ( Card et al, 2013 ; Kasimanickam and Kastelic, 2016 ; Corral-Vazquez et al, 2021 ) and heat shock proteins. The heat shock proteins are a group of tyrosine regulatory elements that participate in hyperactivation and nitric oxide synthesis during fertilization ( Feugang et al, 2010 ; Mohamad et al, 2018 ; Nicolas et al, 2020 ; Lian et al, 2021 ; Sun et al, 2021 ).…”