Developmental RNA-Seq transcriptomics of haploid germ cells and spermatozoa uncovers novel pathways associated with teleost spermiogenesis

Abstract

In non-mammalian vertebrates, the molecular mechanisms involved in the transformation of haploid germ cells (HGCs) into spermatozoa (spermiogenesis) are largely unknown. Here, we investigated this process in the marine teleost gilthead seabream (Sparus aurata) through the examination of the changes in the transcriptome between cell-sorted HGCs and ejaculated sperm (SPZEJ). Samples were collected under strict quality controls employing immunofluorescence microscopy as well as by determining the sperm motion kinematic parameters by computer-assisted sperm analysis. Deep sequencing by RNA-seq identified a total of 7286 differentially expressed genes (DEGs) (p-value < 0.01) between both cell types, of which nearly half were upregulated in SPZEJ compared to HCGs. In addition, approximately 9000 long non-coding RNAs (lncRNAs) were found, of which 56% were accumulated or emerged de novo in SPZEJ. The upregulated transcripts are involved in transcriptional and translational regulation, chromatin and cytoskeleton organization, metabolic processes such as glycolysis and oxidative phosphorylation, and also include a number of ion and water channels, exchangers, transporters and receptors. Pathway analysis conducted on DEGs identified 37 different signaling pathways enriched in SPZEJ, including 13 receptor pathways, from which the most predominant correspond to the chemokine and cytokine, gonadotropin-releasing hormone receptor and platelet derived growth factor signaling pathways. Our data provide new insight into the mRNA and lncRNA cargos of teleost spermatozoa and uncover the possible involvement of novel endocrine mechanisms during the differentiation and maturation of spermatozoa.

Role of Ion Channels in the Maintenance of Sperm Motility and Swimming Behavior in a Marine Teleost

“…The results of the previous experiments suggest the presence of Ca 2+ and K + channels in seabream spermatozoa that may play a role in the maintenance of flagellar motility. To investigate this, we carried out a meta-analysis of the data from a recent RNA-seq transcriptomic study during seabream spermiogenesis [ 30 ] in order to identify which types of ion channels are potentially expressed in the ejaculated spermatozoon. The results of this analysis revealed that seabream spermatozoa express 342 ion channel-encoding genes belonging to 38 different families of ion channels, the groups of voltage-gated K + channels (VGCCs) and voltage-gated Ca 2+ channels (VGKCs) being the most represented ( Figure 5 a, Supplementary Spreadsheet S1 ).…”

“…The transcripts from the seabream ejaculated spermatozoa transcriptome annotated as ion channels or transporters [ 30 ] were classified into different families according to the International Union of Basic and Clinical Pharmacology (IUPHAR)/British Pharmacological Society (BPS) Guide to PHARMACOLOGY ( ; accessed on 1 January 2021) [ 60 ]. The putative subcellular localization of each channel was determined using the Uniprot database ( ; accessed on 1 January 2021).…”

“…The expression levels of each transcript were estimated as FPKMs using the RSEM v1.3.0 [ 61 ] software. The details of RNA-seq library construction, sequencing and annotation are provided in Castro-Arnau et al [ 30 ].…”

“…Data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. Data in a and b were calculated from a recent RNA-seq analysis on seabream spermatozoa reported by Castro-Arnau et al [ 30 ] (Gene Expression Omnibus database accession no. GSE173088, National Center for Biotechnology Information).…”

“…The seabream RNA-seq dataset analyzed in this study is available at the Gene Expression Omnibus (GEO) database from the National Center for Biotechnology Information (NCBI) under accession no. GSE173088 [ 30 ]. All other data are available from the corresponding author upon request.…”