P-745 Follicular fluid exposure may activate gene expression in mature human sperm before fertilization
Authors: Turunen T , Malinen M , Heikkinen S , Heinäniemi M , Swanson G , Krawetz S , Allinen P , Kuusipalo L , Kekäläinen J 
Published: June 2023

Abstract

Study question
Does follicular fluid (FF) exposure shape human sperm RNA content and are these changes acquired through genomic or nongenomic manner?

Summary answer
FF exposure altered sperm RNA content potentially via activation of sperm RNA transcription machinery and caused selective intake of FF RNA transcripts in sperm.

What is known already
Mature sperm have been widely recognized as transcriptionally inert. Therefore, sperm RNAs have been regarded as the sole remnants of the gene expression process that occurs during spermatogenesis. Supporting this view, most sperm histones are replaced by protamines in developing spermatids to enable tighter packaging of the genetic material, which should prevent mature sperm cells to express their genes. However, in humans, approximately 15% of the sperm DNA remains associated with histones, raising the possibility that these genome regions could be available for de novo RNA transcription under an appropriate stimulus.

Study design, size, duration
Fresh, mature sperm were divided into three aliquots. One aliquot was used as an untreated control sample, the second and third aliquots were treated with either PureSperm® Wash (SW) solution or an FF pool of five females for three hours. RNA sequencing (RNA-seq) data was collected from FF and sperm samples. Results were complemented with Chromatin Immunoprecipitation Sequencing (ChIP-seq) data from human mature sperm (GEO: GSE15690 and GSE49624) and nascent RNA capture followed by PCR.

Participants/materials, setting, methods
All male and female participants were of reproductive age, non-smoking, healthy individuals who reported no history of infertility. Differentially expressed genes (DEG) were identified based on RNA-seq data (adjusted p-value &lt; 0.05, logFC &lt; |0.75|). The histone status of the genomic loci of DEGs was evaluated using ChIP-seq data. DEGs were also subjected to Gene Ontology analysis. Activated RNA transcription was confirmed by the Click-iT™ Nascent RNA Capture kit and PCR.

Main results and the role of chance
RNA-seq showed 193 DEGs between untreated and FF-treated samples of which 30 genes were upregulated and 163 were downregulated. Furthermore, 18 DEGs were detected between SW- and FF-treated sperm of which 17 genes were upregulated and one downregulated. Finally, 137 DEGs (all downregulated) were found between untreated and SW-treated samples. Among upregulated genes, chemokine-signaling, ion transport, and steroidogenesis-related biological functions were enriched whereas RNA processing, translation, and protein targeting-related biological processes were enriched among downregulated genes (untreated vs. FF-treated). ChIP-seq data revealed that 93% of upregulated DEGs are located within histone-bound chromatin and most of them bear both active and inactive histone markers. Finally, we confirmed that the upregulation of at least two genes (CXCL3 and CXCL8) resulted from active RNA transcription by sperm, whereas the upregulation of IGF2 was due to the selective internalization of mRNA from FF. Additionally, we identified 1396 protein-coding mRNAs present in human FF that were associated with gene expression-related biological processes such as the regulation of transcription, processing of RNA subtypes, and translation.

Limitations, reasons for caution
Due to the limited sample size, the generalizability of our results should be validated in the future. Furthermore, molecular mechanisms behind observed nascent RNA production needs to be clarified in more detail, including potential FF-mediated remodeling of sperm histone markers involved in the activation of gene expression.

Wider implications of the findings
Our findings demonstrate that FF exposure changes sperm RNA content, which seems to be partly caused by the active transcription of mature sperm, which has been widely believed to be non-existent. Therefore, our results can have many important implications for a deeper understanding of fertilization, early embryogenesis, and infertility.

Trial registration number
Not applicaple