Protamine lacking piscine spermatozoa are transcriptionally active

Transcriptional quiescence of post-meiotic spermatozoa associated with protamine-mediated chromatin condensation is widely recognized in animals. How sperm acquire the extratesticular maturational competence to move and fertilize the egg is therefore thought to occur via non-transcriptional mechanisms. Here, using transcriptional profiling during spermatozoon differentiation in a fish that does not condense chromatin with protamines, we uncover spatially distinct roles of the GnRH receptor and PDGF signaling pathways between the somatic epithelia of the extratesticular ducts and the maturing spermatozoa. In vitro induction and inhibition experiments demonstrate that the endocrine signaling pathways are conserved in different lineages of fish and activate de novo transcription of spermatozoon genes required for the acquisition of full motility. These experiments further confirmed that mitochondrial translation is important for sperm maturation in anamniotes as in amniotes, but that transcriptional quiescence of post-meiotic spermatozoa is not a pan vertebrate phenomenon. On the contrary, the data show that the identified signal transduction pathways between the soma and the sperm upregulate effector genes essential for maturational competence and male fertility.

By contrast, almost nothing is known of the molecular signaling pathways that regulate 60 sperm maturation in anamniotes. formed by spermatocytes II and spermatids (SPC II and SPD, respectively), which we refer 125 here as HGCs, and another subpopulation formed by intratesticular spermatozoa (SPZ I ) 126 ( Figure 1A and B). The percentage of HGCs and SPZ I in the testicular extracts was of 34 ± 127 4% and 66 ± 4% (n = 15), respectively ( Figure 1B). 128 Microscopic examination of the HGC-enriched population after FACS confirmed the 129 presence of SPC II, and round and elongating SPD in this fraction ( Figure 1C). Whole-130 mount immunostaining revealed strong expression of Lys 9 acetylated histone 3 (H3K9ac) 131 and meiotic recombination protein Spo11 in SPC II, which progressively decreased in 132 6 round and elongating SPD, and completely vanished in SPZ EJ (Figure 1C). 133 Immunolocalization of α-tubulin (Tuba) showed that the protein was spread in the 134 cytoplasm in SPC II and round SPD, became also detectable in the nascent flagellar region 135 of elongating SPD, and was finally distributed along the flagellum of differentiated SPZ EJ 136 ( Figure 1C). These observations indicate a high occurrence of meiotic recombination in 137 SPC II and a progressive DNA condensation during the differentiation of SPC II into SPD 138 and spermatozoa, which are conserved features during vertebrate germ cell development 139 (Kurtz et al., 2009; Hazzouri et al., 2000). Therefore, these data confirmed that the sorted  In total, 31,501 protein-coding genes were annotated, which produced 57,396 transcripts   (Figure 1G). 166 The quality of the RNA-seq data and the reliability of the DEGs identified were  (Figure 2F). 216 In an effort to identify specific transcription/translation and metabolic processes 217 enriched in SPZ EJ , we built the protein interactome network of DEGs classified into these 218 two categories by using the STRING protein-protein interaction (PPI) database for known    Figure 5D). 300 The cell localization of pdgf expression in testis and ETDs by in situ hybridization  (Figure 6B and C). In contrast, pdgfd expression was low in the 312 epithelium throughout the ETDs, but somewhat more intense in the proximal region of the 313 SD ( Figure 6D), while the expression of pdgfaa and -c was almost or completely 314 undetectable (Figure 6-Supplement 1). 315 Taken together, these findings demonstrate the local production of Gnrh peptides by  Figure 7A). These data 327 therefore indicate that SPZ ED can be classified as immature gametes, which acquire full 328 motility potential during their journey throughout the ETDs.

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Further RT-PCR analysis showed that both SPZ ED and SPZ EJ express gnrhr1, gnrhr2 330 and gnrhr3 transcripts (Figure 7B), while expression of pdgfra is specific of SPZ ED , and 331 that of pdgfrb is prevalent in SPZ ED and SPZ EJ ( Figure 7C). Therefore, we tested the 332 hypothesis that the activation of these receptors by their cognate ligands in SPZ ED may play   Figure 8B-D), thus suggesting the existence of the epithelial GnRH and PDGF signaling 382 pathways in the ETD of zebrafish as observed in seabream.

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To further assess whether sbGnRH and rPDGF-BB can induce sperm maturation in 384 zebrafish, we first confirmed that spermatozoa from the ETD (SPZ ETD ) show lower motility 385 than SPZ EJ upon activation in freshwater (Figure 8-Supplement 2), and that they express  In most teleosts, sperm maturation, the phase during which non-functional gametes 408 develop into mature spermatozoa, fully capable of vigorous motility and fertilization, is 409 believed to occur in the ETDs (Schulz et al., 2010). Previous studies have shown that 410 administration of some hormones, such as progestins, androgens and gonadotropins, can 411 increase the seminal plasma pH in the ETD, which results in the elevation of intra-sperm 412 cAMP levels, increase hydration, or induce the secretion of sperm-immobilizing ions by the 413 ETD epithelium (Schulz et al., 2010; Marshall et al., 1993). However, the cellular sources  which is an axonemal protein that plays a role in PKA-dependent signaling cascades 473 required for spermatozoon capacitation (Fiedler at al., 2013). These findings therefore 474 reinforce the notion that GnRH and PDGF signaling from the ETD epithelium plays a 475 paracrine role to specifically induce the maturational expression of genes required for the 476 activation and prolongation of sperm motility in the external aquatic environment.  (Kurtz et al., 2009; Saperas et al., 1993). In such cases, and indeed in a highly 489 diverse range of species, including invertebrates, the first chromatin condensation transition 490 is also characterized by low level acetylation that is not related to histone replacement 491 (Kurtz et al., 2007, 2009). It seems plausible that the de novo transcription observed for the 492 maturing spermatozoa of seabream in the present study, may therefore occur during this 493 phase. In any event, the regional signaling of the ETD appears to be conserved in the Spain) and maintained in the laboratory as previously described (Chauvigné et al., 2013). 513 Samples of testis and SPZ EJ were obtained from males during the natural reproductive   Nextera® XT (Illumina) and amplified for 12 cycles with indexed Nextera® PCR primers.

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The library was purified twice with Agencourt Ampure XP beads (0.8:1 ratio) and 571 quantified on a Bioanalyzer using a High Sensitvity DNA Kit.

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The libraries were sequenced on HiSeq2500 (Illumina) in paired-end mode with a 573 read length of 2 x 76bp using TruSeq SBS Kit v4. We generated more than 30 million To improve the gilthead seabream reference genome (Pauletto et al., 2018) for the 581 differential expression analysis, the genome was reannotated, and a de novo transcriptome 582 assembly was generated from which those transcripts not present in the genome assembly 583 were added to the analysis. The gene predictors were run with trained parameters for human except Genemark that runs 598 on a self-trained manner. Finally, all the data was combined into consensus CDS models 599 using EvidenceModeler-1. 1.1 (Haas et al., 2008). Additionally, UTRs and alternative 600 splicing forms were annotated through two rounds of PASA annotation updates. Functional 601 annotation was performed on the annotated proteins with Blast2go (Conesa et al., 2005), 602 using Blastp (Altschul et al., 1990) (Haas et al., 2013) and functional annotation was performed on the annotated proteins with 627 22 Blast2GO, as described above. Finally, the assembled transcripts were mapped against the 628 seabrem reference genome assembly with GMAP (Wu et al., 2005). Those transcripts for 629 which less than 50% of their length aligned to the genome, and with a complete ORF and 630 functional annotation, were added to the reference genome as separate annotated contigs. The GO enrichment of DEGs and signaling pathway analyses were performed using the 646 PANTHER v14.1 Classification System and analysis tools (http://www.pantherdb.org/).

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GO terms and pathways with FDR < 0.05% were considered significant. Scattered plot of 648 pathway analysis was carried out with 'ggplot2' R package. Functional categories 649 classification were also done manually using the Uniprot database 650 (https://www.uniprot.org/) and QuickGO browser (http://www.ebi.ac.uk/QuickGO).