Single-Cell Transcriptomics reveals relaxed evolutionary constraint of spermatogenesis in two passerine birds as compared to mammals

Abstract

Spermatogenesis is a complex process where spermatogonia develop into haploid, mobile sperm cells. The genes guiding this process are subject to an evolutionary trade-off between preserving basic functions of sperm while acquiring new traits ensuring advantages in competition over fertilization of female gametes. In species with XY sex chromosomes, the outcome of this trade-off is found to vary across the stages of spermatogenesis but remains unexplored for species with ZW sex chromosomes. Here we characterize avian spermatogenesis at single cell resolution from testis of collared and pied flycatchers. We find evidence for relaxed evolutionary constraint of genes expressed in spermatocyte cells going through meiosis. An overrepresentation of Z-linked differentially expressed genes between the two species at this stage suggests that this relaxed constraint is associated with the lack of sex-chromosome silencing during meiosis. We conclude that the high throughput of bird spermatogenesis, at least partly, is explained by relaxed developmental constraint.

The genomic basis of hybrid male sterility in Ficedula flycatchers

“…Our results may appear contractionary in the light of the first developmental stages of spermatogenesis (i.e. mitosis and meiosis) being known to be strongly evolutionary constrained both in mammals (Larson et al, 2018;Kopania et al, 2022) and in birds (Segami et al 2022). Most of the known examples of genes associated with hybrid sterility in animals are, in fact, expressed during either the pre-or post-meiotic stages of gametogenesis (Coyne & Orr, 2004).…”

“…We created a consensus clustering using the 6 pure species samples to use as a baseline for all our consecutive analysis. The identities of the obtained 17 different cell clusters (Figure 1A) were verified using the characterized flycatcher markers for spermatogenesis populations developed by Segami et al 2022 (Figure S1). Our new clusters corresponded to all the previously identified testis cell clusters in flycatcher spermatogenesis except for one of the somatic cell clusters that was not found (Segami et al 2022).…”

“…For all the 4 clusterings we found an heterogeneous cluster with no good markers that we excluded and reclustered the remaining cells again. After obtaining all our final clusterings we established the identity of all clusters using the flycatcher gene markers characterized by (Segami et al 2022). We then established the equivalences of the clusters across our different clusterings (Figure 4).…”

“…Mugal et al, 2020 found an intermediate gene expression of testis genes in bulk samples from hybrids indicating no major incompatibilities between collared and pied flycatchers. However, hybrids may have a different testis cell composition than the pure species (Hunnicutt et al, 2022) and we therefore recently characterized the spermatogenesis of Ficedula flycatchers at a single cell level to reveal species differences in gene expression at specific stages of the process (Segami et al, 2022). We found that most differentially expressed (DE) genes were active after meiosis and typically coding for cell respiratory or motility pathways at a late stage of spermatogenesis.…”

“…Here, we aim at identifying the molecular basis of sterility in Ficedula male hybrids by characterizing the hybrid spermatogenesis transcriptome at a single cell level. Using single-cell RNA sequencing of testis cell suspensions of two hybrids, three Collareds and three Pied flycatchers and markers developed by (Segami et al, 2022) and histology sections we first aim to investigate whether hybrid males go through all “normal” stages of spermatogenesis by identifying if hybrids have all testis cell types found in the parental species. Secondly, we will investigate whether hybrid expression patterns are different from the parental species in any particular stage of spermatogenesis as an indication of a possible failure.…”