Preliminary Studies On The Role Of Heat Shock Transcription Factor 5 In The Process Of Spermatogenesis

Background and ObjectiveInfertility is a global health problem.Statistically,approximate 1 out of 7 couples at reproduction age is unable to be pregnant.Among these infertile couples,male infertility accounts for about 30%.Infertility not only exposes the couples who intend to bear children to heavy psychological burden,but also brings pressure on social medical services.Most male infertility patients exhibit a decrease in sperm quality,but the etiology of this symptom is unclear,and as a consequence,there is no effective treatment for most male infertility cases.In recent years,the role of environmental heat stress elements in male infertility has attracted much attention.As an important transcription factor family in eukaryotes,heat shock transcription factors(HSFs)are key regulators in cellular stress,differentiation,tissue development and organismal reproduction.HSFs regulate a plethora of genes including those encoding heat shock proteins(HSPs)by recognizing and binding to heat shock elements(HSEs)within the promoter of target genes.Heat shock transcription factor 5(HSF5)is one of the six HSFs encoded in human genome,but its physiological functions and expression profiles remain to be determined.A recent study found that male zebrafish were be sterile upon deletion of the hsf5 gene deletion,which broadens our ken on etiology,prevention and therapy of male infertility.This study aimed to characterize HSF5,detect possible target genes and explore its potential function in germ cell development using technologies of structural biology,gene knockdown and RNA sequencing,and thus to provide a new direction for the diagnosis and therapy of male infertility.Methods1.HSF5 protein expression plasmids carrying an N-terminal His-tag were constructed after sequence analysis and comparison,selection of target genes and plasmid construction.The recombinant protein was expressed in E.coli,and purified using nickel ion affinity chromatography and gel filtration chromatography.The DNA-binding activity of the purified protein was detected by running a DNA fragment containing HSE motifs in agarose gel,which might be migrated by HSF5-binding.2.Crystallization of recombinant human HSF5 in complex with DNA were screened,and the established conditions were subsequently optimized.The obtained HSF5-DNA complex crystals were taken to the Shanghai Synchrotron Light Source Centre for X-ray diffraction,where data collection was done.After data processing,the structure of the HSF5-DNA complex was determined by molecular replacement using HSF2 as a search model.3.The Hsf5 gene was knocked down by siRNA interference in murine spermatocytes(GC-2,BALB/c mice),the outcome of which was verified by qPCR.Transcriptome sequencing was conducted using the same cells without siRNA interference as a reference and verify the result preliminarily.Results1.The HSF5-expression plasmid carrying a His-tag purification tag was successfully constructed.A highly pure and soluble recombinant human-derived HSF5 protein was successfully produced in the prokaryotic system,and the molecular weight of the target protein was verified to be consistent with the theoretical protein molecular weight by SDSPAGE and Western bolt.The purity of the target protein was above 90% at a concentration of about roughly 40 mg/m L,and its biochemical activity was verified by gel migration assays,which showed a bond of HSEs-containing oligonucleotides.2.Four preliminary crystallization conditions were selected for optimization.Large,regular-edged single crystals were grown under the optimal conditions,which were subjected to X-ray diffraction,with data collected at synchrotron X-ray source and processed using XDS.The structure of the HSF5-DNA complex was solved at 2.9 (?).3.The success of the siRNA interference experiments was verified by qPCR,and the transcriptome of murine spermatocytes after the interference was sequenced.Murine spermatocytes with Hsf5 knockdown were found to exhibit down-regulation of immunerelated functions.The successful inflammatory response model of lipopolysaccharide revealed that compared with lipopolysaccharide control group,the levels of IL-1β and IL-6 in cell supernatant were decreased in the lipopolysaccharide Hsf5-knockdown group.ConclusionsThe recombinant human HSF5 protein was expressed in soluble form using the prokaryotic system,and was biochemically active.The structure of HSF5 shows a typical winged helix-turn-helix core,with its third helix(α3)inserted into the large groove of DNA.A highly conserved arginine residue in HSF5,Arg82,forms a specific bidentate hydrogen bonds with a highly conserved guanine in HSEs,thus establishing a readout mechanism utilized by HSF5.Notably,the second helix(α2)of HSF5 is interrupted by a longer flexible loop,which extend away from the DNA binding surface and seems probably to participate in protein-protein interactions.Transcriptome sequencing revealed that Hsf5 gene knockdown in murine spermatocytes likely leads to down-regulation of immune-related functions,suggesting that HSF5 may act as an immune protector in mouse germ cells.This study has laid the foundation for uncovering the relationship between HSF5 and male reproduction.