Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice.

The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular-based protein delivery platforms transport proteins to the developing head and tail: the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review, we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra-manchette transport to the basal body at the base of the developing sperm tail and through the intraflagellar transport to the construction site in the flagellum. This review focuses on the microtubule-based mechanisms involved and the consequences of their disruption in spermatid elongation.Reproduction (2016) 151 R43-R54

Male infertility is an increasing problem partly due to inherited genetic variations. Mutations in genes involved in formation of the sperm tail cause motility defects and thus male infertility. Therefore, it is crucial to understand the protein networks required for sperm differentiation. Sperm motility is produced through activation of the sperm flagellum, which core structure, the axoneme, resembles motile cilia. In addition to this, cytoskeletal axonemal structure sperm tail motility requires various accessory structures. These structures are important for the integrity of the long tail, sperm capacitation, and generation of energy during sperm passage to fertilize the oocyte. This review discusses the current knowledge of mechanisms required for formation of the sperm tail structures and their effect on fertility. The recent research based on animal models and genetic variants in relation to sperm tail formation and function provides insights into the events leading to fertile sperm production. Here we compile a view of proteins involved in sperm tail development and summarize the current knowledge of factors contributing to reduced sperm motility, asthenozoospermia, underline the mechanisms which require further research, and discuss related clinical aspects on human male infertility.

Correct gene expression patterns form the basis for male germ cell differentiation and male fertility. Although previous studies have elucidated the importance of testis specific gene expression, the exact transcripts and comprehensive gene expression patterns remain unknown. Large scale sequencing techniques have enabled cost effective analysis of gene expression and isoform studies. Using the SOLiD 4 next-generation sequencing platform we have investigated the gene expression patterns at five different time points during the first wave on murine spermatogenesis. Our results highlight the upregulation of spermatogenesis related biological processes and associated cellular components. Elucidation of differential gene expression at important time points during the sperm development emphasizes the importance of correct timing of gene expression within biological processes. Differential gene level expression was analyzed with R/Bioconductor’s Limma package and isoform analysis was conducted with the Cufflinks pipeline. At gene level total of 2494 differentially expressed genes were identified and Cufflinks characterized over 160 000 gene isoforms, of which 29% were novel transcripts assigned to known genes. Isoforms were detected for 57% of expressed genes and in a total over 26 000 genes were expressed in the testis. Differential promoter and transcription start site usage appears also to play a role in regulation of gene expression during spermatogenesis. Furthermore, we identified 947 upregulated long non-coding RNAs during the first wave of spermatogenesis. These RNAs appeared to be highly specific to different time points. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. Our study provides a very valuable basis for investigation of gene isoforms and regulation and factors contributing to male fertility.

The core axoneme structure of both the motile cilium and sperm tail has the same ultrastructural 9 + 2 microtubular arrangement. Thus, it can be expected that genetic defects in motile cilia also have an effect on sperm tail formation. However, recent studies in human patients, animal models and model organisms have indicated that there are differences in components of specific structures within the cilia and sperm tail axonemes. Primary ciliary dyskinesia (PCD) is a genetic disease with symptoms caused by malfunction of motile cilia such as chronic nasal discharge, ear, nose and chest infections and pulmonary disease (bronchiectasis). Half of the patients also have situs inversus and in many cases male infertility has been reported. PCD genes have a role in motile cilia biogenesis, structure and function. To date mutations in over 40 genes have been identified cause PCD, but the exact effect of these mutations on spermatogenesis is poorly understood. Furthermore, mutations in several additional axonemal genes have recently been identified to cause a sperm-specific phenotype, termed multiple morphological abnormalities of the sperm flagella (MMAF). In this review, we discuss the association of PCD genes and other axonemal genes with male infertility, drawing particular attention to possible differences between their functions in motile cilia and sperm tails.

The immotile short-tail sperm defect is an autosomal recessive disease within the Finnish Yorkshire pig population. This disease specifically affects the axoneme structure of sperm flagella, whereas cilia in other tissues appear unaffected. Recently, the disease locus was mapped to a 3-cM region on porcine chromosome 16. To facilitate identification of candidate genes, we constructed a porcine-human comparative map, which anchored the disease locus to a region on human chromosome 5p13.2 containing eight annotated genes. Sequence analysis of a candidate gene KPL2 revealed the presence of an inserted retrotransposon within an intron. The insertion affects splicing of the KPL2 transcript in two ways; it either causes skipping of the upstream exon, or causes the inclusion of an intronic sequence as well as part of the insertion in the transcript. Both changes alter the reading frame leading to premature termination of translation. Further work revealed that the aberrantly spliced exon is expressed predominantly in testicular tissue, which explains the tissue-specificity of the immotile short-tail sperm defect. These findings show that the KPL2 gene is important for correct axoneme development and provide insight into abnormal sperm development and infertility disorders.cilia ͉ retrotransposon ͉ spermatogenesis

Background.The correct formation of the sperm tail and manchette are essential for male fertility. Sperm tail development is a complex process organized by intraflagellar transport (IFT), mechanism that is utilized to transport molecules along the axonemal microtubule doublets. Two motor proteins are responsible for the transport; kinesin II, the anterograde motor, carries particles from the base towards the site of tail assembly. The retrograde motor, dynein, restores particles and motorproteins back to the pool of IFT components. Manchette is a microtubule and F-actin containing structure expressed transiently during spermiogenesis. It serves as a platform for intramanchette transport (IMT) where particles are carried first from the cytosol or Golgi to the manchette and then IMT delivers proteins to the sperm head or basal body region. We are interest in one of the kinesin II motor protein subunits, KIF3A, which has been shown to be present during sperm tail development, but its specific functions during spermiogenesis are poorly understood. Results.We have localized KIF3A in wild type mice in the manchette, basal body and flagella of elongating spermatids and in the principal piece of mature sperm tail. The depletion of KIF3A results in defects at late spermatogenesis. Spermatogonia, spermatocytes and round spermatids appear normal, but elongating spermatids have short and immotile flagella with disorganized axoneme and accessory structures. Manchette was elongated and perinuclear ring seems to squeeze the developing head causing its knob-like appearance. We were able to identify meiosis-specific nuclear structural protein 1 (MNS1) as an interacting partner for KIF3A. These proteins co-localize in the manchette and principal piece of the sperm tail. MNS1 appears to be delivered through manchette to the sperm tail, where it is required for the assembly of the flagella. In KIF3A KO mice manchette clearance was delayed and MNS1 staining remained in the manchette. Conclusions.Depletion of KIF3A caused defects during sperm tail development, manchette function and head shaping. Its interaction with MNS1 indicates that KIF3A may be involved in the transport of MNS1 to the developing tail. MNS1 concentrates in the manchette in the KIF3A KO suggesting a delay in the transport through the IMT. In addition, MNS1 and KIF3A co-localize in principal piece indicating the possible interaction site in mature sperm. We suggest that KIF3A has a role in manchette formation and IMT in addition to its well defined role in IFT. This study also highlights the essential role of KIF3A and IFT during spermiogenesis.

SPEF2 is expressed in all ciliated cells and is essential for correct sperm tail development and male fertility. We have previously identified a mutation within the SPEF2 gene as the cause for infertility because of immotile and malformed sperm tails in pigs. This mutation in pigs alters the testis-specific long SPEF2 isoform and exclusively affects the sperm tail development. In infertile boars, axonemal and all accessory structures of the sperm tail are affected; thus, SPEF2 seems to participate in the organization of these structures. In the present study, we have investigated the expression of SPEF2 during mouse spermatogenesis. SPEF2 mRNA and protein products appear to be localized both in germ cells and in Sertoli cells. In differentiating germ cells, SPEF2 protein is localized in the Golgi complex, manchette, basal body, and midpiece of the sperm tail. In mature murine sperm, SPEF2 is present in the distal part of the sperm tail midpiece. Using yeast two-hybrid assay and coimmunoprecipitation experiments, we identified an interaction between SPEF2 and the intraflagellar transport protein IFT20 in the testis. Furthermore, these two proteins colocalize in differentiating male germ cells. These results support the crucial importance of SPEF2 in sperm differentiation and involvement of SPEF2 in structuring of the sperm tail.