Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model

Abstract

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) are an ancestral eukaryotic trait that has been lost in several lineages like flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) ecotypes to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute ecotype to identify a set of candidate genes associated with moss male infertility. We generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Defects in mammal and algal homologues of this gene coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. The Ppccdc39 mutant resembles the Gransden phenotype in terms of male fertility. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of Gransden, causing regulatory differences of for example the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility defect. We propose that moss spermatozoids might be employed as an easily accessible system to study male infertility of humans and animals in terms of flagellar structure and movement.

Keywords: Physcomitrella patens; cilia; flagella; male infertility; moss; sperm; spermatozoid.

Figure 1:. Cladogram displaying independent losses of motile sperm during plant evolution and Physcomitrella patens sexual life cycle.

(a): The LECA (last eukaryotic common ancestor) gave rise to eukaryotes of all five kingdoms, in which Archaeplastida harbour the green lineage. Zygnematophyceae, Coleochaetophyceae and Charophyceae (ZCC grade) are sister to land plants (One Thousand Plant Transcriptomes, 2019). Bryophytes are probably monophyletic (see text for references) comprise hornworts, liverworts and mosses, and are sister to vascular plants. Flagella were present in the LECA and were lost three times independently during plant evolution (red cross indicates absence of flagella). Drawn based on DeVries and Archibald 2018, Mast et al., 2014, Puttick et al., 2018. (b): Upon environmental stimulus, reproductive organs (gametangia) develop on the apex of each gametophore, namely archegonia (female) and antheridia (male). Additionally, paraphyses emerge (p). Mature antheridia release the motile (flagellated) spermatozoids upon watering. The sperm cells swim through the archegonial venter to fertilize the egg cell. After fertilization, embryo development (E1/E2) and sporophyte development (ES-B) occurs. The mature sporophyte is located on the apex of the gametophore and releases haploid spores of the next generation. P. patens is predominantly selfing. Embryo/sporophyte developmental stages according to Hiss et al. (2017).

Figure 2:. Comparative analysis of Physcomitrella patens ecotypes Gransden (Gd) and Reute (Re) male reproductive apparatus.

(a): Crossing analysis between Re (n = 430), Gd (n = 300) and fluorescent marker line Re-mcherry. Re develops 98.1% of sporophytes per gametophore of which 3.4% are heterozygous (derived from crossing, not selfing). Gd develops 88.33% sporophytes per gametophore of which 97.3% are heterozygous. Mean represented by black line. Significance shown by asterisk (Chi-square test, p < 0.01, four biological replicates each). (b): Re (n = 10) and Gd (n = 3) spermatozoids differ significantly in velocity (two-sided t-test, p < 0.01,*). Mean represented by black line. Note that only three Gd spermatozoids were measured because the majority shows no motility at all (Fig. S6B), and hence velocity = 0. (c): Scanning electron micrograph (SEM) of Re sperm cell showing cell architecture. From the cell anterior (a), the two flagella emerge (arrow) at staggered locations from the coiled nucleus (n). One of two long flagella (f) is visible and extends beyond the cell proper. (d): Phase contrast image of swimming Re spermatozoids. (e): Transmission electron micrograph (TEM) cross-sections of mature Gd flagella showing cytoplasmic connections (cc) that connect coils. (f): TEM of a mature spermatozoid of Re. The axoneme exhibits clear outer dynein arms (black arrows) and the plasmalemma is closely associated with the nine doublets. (g): SEM of Gd spermatozoid with architecture as in Re spermatozoids in C, except the flagella remain coiled. (h): Phase contrast image of swimming Gd spermatozoids showing loops at the posterior end of the flagella (arrow). (i): TEM higher magnification of a Gd flagellum in figure 2E showing fewer electron densities associated with the central pair complex (arrow) and microtubule doublets.

Figure 3:. Physcomitrella patens bell1 and ccdc39 expression, GO bias of DMP/SNP overlap and ccdc39 phylogeny.

(a): Gene Ontology bias analysis of genes differentially methylated and SNP- containing in comparison of Gd vs. Re. The intersect of DMP and SNP affected genes shows over-representation of GO terms associated with cilia motility. Over-represented terms are shown in green, whereas under-represented terms are shown in red. Larger font size correlates with a higher significance level. (b): RT-PCR expression analysis of genes expressed in Gd and Re adult apices shows expression of bell1 (314 nt) in the Re (white arrow) but not in the Gd background. (c): Relative expression of ccdc39 in qPCR (green, n = 3) and RNA-seq (blue, n = 3) data shows higher expression in Re (mean qPCR: 1, mean RNA-seq: 1) in comparison to Gd (mean qPCR: 0.6, mean RNA-seq: 0.89). Mean marked by black bars. (d): Phylogenetic analysis of CCDC39 shows clear correlation with the presence of flagella and generally reflects species relationships. Planta are shown in green, opisthokonts in yellow, protozoa, SAR and fungi in black. Homo sapiens, Chlamydomonas reinhardtii and Physcomitrella patens are marked in red.

Figure 4:. Analysis of Physcomitrella patens ccdc39.

(a): Selfing and crossing analysis between ccdc39 (n = 630), Reute (Re) (n = 331) and fluorescent marker line Re-mcherry. ccdc39 develops 0% and Re develops 98.6% sporophytes per gametophore under selfing conditions. Under crossing conditions, Re develops 98.1% of sporophytes per gametophore of which 2,8% are crosses. ccdc39 develops 94.1% sporophytes per gametophore of which 100% are crosses (asterisk shows significant deviation, Chi-square test, p < 0.01). Mean marked by black line. Re (b) and ccdc39 (c) apices 30 days after watering show properly developed sporophytes on Re apices and bundles of gametangia on ccdc39 apices. d: Phase contrast image of fixed mature ccdc39 spermatozoids displaying posterior flagellar loops (arrows). (e): SEM of ccdc39 spermatozoid that remains in coils. The nucleus (n) is a long coiled cylinder that narrows posteriorly. Irregular flagella coil around the cell (arrowhead). (f): TEM of ccdc39 whole mature spermatozoid in cross section with compacted nucleus (n) in upper coil. Arrowheads indicate irregular flagella that encircle the cell for over 2 revolutions. Cytoplasmic connections (cc) adjoin axonemes across their length. Bar = 500 nm. (g): TEM cross-section of a Re immature axoneme showing the regular pattern of nine outer microtubule doublets and two single microtubulis in the central pair complex (white arrow) as well as outer dynein arms (od, black arrows). (h-j): TEM cross-sections of irregular ccdc39 flagella showing intact acentric central pair complexes (white arrowheads), outer dynein arms (od, black arrows). (h, j): Double axonemes in single flagellar shafts can be observed.