RNA Interference-Mediated Downregulation of sAC Gene Inhibits Sperm Hyperactivation in Male Rats (Rattus norvegicus)

Abstract

Hyperactivation is one of the most critical parts for fertilization. cAMP generated by soluble adenylyl cyclase (sAC) is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and were separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL (Curvilinear velocity) and ALH (Amplitude of lateral head displacement) of RNAi transfected sperm were reduced. cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.

Transcriptome Sequencing Reveals the Differentially Expressed lncRNAs and mRNAs Involved in Cryoinjuries in Frozen-Thawed Giant Panda (Ailuropoda melanoleuca) Sperm

“…It has also been shown that ejaculated sperms can translate protein from mRNA transcripts during the final maturation steps prior to fertilization. Besides, knocking down the hyperactivation-associated mRNA by transfecting siRNA can inhibit the level of cAMP and protein oxidative phosphorylation in spermatozoa and reduce the level of sperm hyperactivation [ 15 , 16 ]. Recently, microRNA (miRNA) and long non-coding RNA (lncRNA) have been demonstrated to be involved in spermatogenesis [ 17 ], sperm cryoinjuries [ 18 ] and fertility [ 19 ].…”

Systematic identification and characterization of long non-coding RNAs in mouse mature sperm

“…The knockdown of coding genes retained in sperm could impair the sperm function [34, 35]. Hence, the putative functional lncRNAs that affect nucleic acid metabolic processes might affect the spermatogenesis or mature sperm function via modulating corresponding target genes.…”

“…Sperm might retain certain transcriptional and translational activities [ 32 , 33 ], which might result in the intact RNAs function in regulating targeted gene expression in sperm. The knockdown of coding genes retained in sperm could impair the sperm function [ 34 , 35 ]. Hence, the putative functional lncRNAs that affect nucleic acid metabolic processes might affect the spermatogenesis or mature sperm function via modulating corresponding target genes.…”

Knockdown of sAC affects sperm hyperactivation by cAMP-signaling pathway in male rat (Rattus norvegicus)

“…The in vitro experiments results showed that shRNA717 and shRNA4205 were both optimal target sequences with the highest knockdown efficiency [26]; therefore, shRNA717 and shRNA4205 were chosen as the targeted sequence sites for plasmid construction. We designed two sAC shRNA oligonucleotides for this experiment ( Table 1).…”

“…Lactate dehydrogenase C (LDHC) plays an important role in the maintenance of glycolysis [35]. In this process, CO 2 [26]. Reducing the sAC protein amount decreases the use of ATP and Ca 2+ and then CatSper2 and LDHC expressions are suppressed.…”