Characterization 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) with flagella are an ancestral eukaryotic trait that has been lost in several lineages, for example in flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) strains to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute strain to identify a set of genes associated with moss male infertility. Defects in mammal and algal homologs of these genes coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. As a proof of principle, we generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Indeed, the Ppccdc39 mutant resembles the male infertile Gransden strain phenotype. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of P. patens Gransden, causing regulatory differences of e.g. the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility. We propose that P. patens spermatozoids might be employed as an easily accessible system to study male infertility of human and animals.

DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth

“…In both cases dek1‐tomato int remained sporophyte‐less, indicating that this specific tagging of DEK1 has an effect on the fertility of the female germ line, or on activation of the zygote program. When isolated (Meyberg et al , ), dek1‐tomato int and Gransden spermatozoids did not show any significant morphological differences (Fig. S5).…”

“…However, the partially developed spermatozoid appeared more numerous than in the WT strain, potentially indicating that the fluorescent tag may negatively affect spermatogenesis. Testing for male fertility is more challenging, but the recent establishment of a male‐specific sterile mutant ccdc39 , without other apparent defects (Meyberg et al , ), allowed us to test dek1‐tomato int for male fertility. To this effect, we performed a standard crossing assay between dek1‐tomato int and ccdc39 in parallel with the control cross Gransden and ccdc39 .…”

“…At the phenotypic level, the strong signal during spermatogenesis may hint at the cause of the male sterility observed in crossing trial. We ruled out that the absence of a sporophyte could be attributed to the well‐documented low male fertility of the Gransden strain in general (Perroud et al , , ; Landberg et al , ; Ashton & Raju, ; Hiss et al , ; Meyberg et al , ). However, it is possible that vegetative amplification of dek1‐tomato int for several years led to a sterility problem.…”

“…Gametogenesis and sporophyte culture were performed as described previously (Perroud et al , ). Antheridium and spermatozoid isolation were performed as described previously (Meyberg et al , ) between 21 to 30 d after juvenile gametophore transfer to 15°C, with a photoperiod of 8 h: 16 h, light (20 μmol s − 1 m −1 ) : dark.…”

“…Spermatozoids were isolated from a mature antheridium as described previously (Meyberg et al , ). They were dried rapidly, fixed on a glass slide and subsequently stained with DAPI before being mounted for microscopy observation.…”