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.
Sperm differentiation requires specific protein transport for correct sperm tail formation and head shaping. A transient microtubular structure, the manchette, appears around the differentiating ...spermatid head and serves as a platform for protein transport to the growing tail. Sperm flagellar 2 (SPEF2) is known to be essential for sperm tail development. In this study we investigated the function of SPEF2 during spermatogenesis using a male germ cell-specific
knockout mouse model. In addition to defects in sperm tail development, we observed a duplication of the basal body and failure in manchette migration resulting in an abnormal head shape. We identified cytoplasmic dynein 1 and GOLGA3 as novel interaction partners for SPEF2. SPEF2 and dynein 1 colocalize in the manchette and the inhibition of dynein 1 disrupts the localization of SPEF2 to the manchette. Furthermore, the transport of a known SPEF2-binding protein, IFT20, from the Golgi complex to the manchette was delayed in the absence of SPEF2. These data indicate a possible novel role of SPEF2 as a linker protein for dynein 1-mediated cargo transport along microtubules.
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.
Primary ciliary dyskinesia (PCD), a disease caused by the malfunction of motile cilia, manifests mainly with chronic recurrent respiratory infections. In men, PCD is also often associated with ...infertility due to immotile sperm. Since causative mutations for PCD were identified in over 50 genes, the role of these genes in sperm development should be investigated in order to understand the effect of PCD mutations on male fertility. Previous studies showed that different dynein arm heavy chains are present in respiratory cilia and sperm flagellum, which may partially explain the variable effects of mutations on airways and fertility. Furthermore, recent studies showed that male reproductive tract motile cilia may play an important part in sperm maturation and transport. In some PCD patients, extremely low sperm counts were reported, which may be due to motile cilia dysfunction in the reproductive tract rather than problems with sperm development. However, the exact roles of PCD genes in male fertility require additional studies, as do the treatment options. In this review, we discuss the diagnostic and treatment options for men with PCD based on the current knowledge.
Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused ...by malfunction of motile cilia, hair-like organelles protruding out of the cell that are responsible for removal of mucus from the airways and organizing internal organ positioning during embryonic development. PCD is caused by mutations in genes coding for structural or assembly proteins in motile cilia. Thus far mutations in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported, thus highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics. In this study, we identified a recurrent loss-of-function mutation c.198_200delinsCC in
CFAP300
causing lack of the protein product. PCD patients homozygous for the identified
CFAP300
mutation have immotile airway epithelial cilia associated with missing dynein arms in their ciliary axonemes. Furthermore, using super resolution microscopy we demonstrate that CFAP300 is transported along cilia in normal human airway epithelial cells suggesting a role for CFAP300 in dynein complex transport in addition to preassembly in the cytoplasm. Our results highlight the importance of CFAP300 in dynein arm assembly and improve diagnostics of PCD in Finland.
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.
Cattle populations are highly amenable to the genetic mapping of male reproductive traits because longitudinal data on ejaculate quality and dense microarray-derived genotypes are available for ...thousands of artificial insemination bulls. Two young Nordic Red bulls delivered sperm with low progressive motility (i.e., asthenospermia) during a semen collection period of more than four months. The bulls were related through a common ancestor on both their paternal and maternal ancestry. Thus, a recessive mode of inheritance of asthenospermia was suspected.
Both bulls were genotyped at 54,001 SNPs using the Illumina BovineSNP50 Bead chip. A scan for autozygosity revealed that they were identical by descent for a 2.98 Mb segment located on bovine chromosome 25. This haplotype was not found in the homozygous state in 8557 fertile bulls although five homozygous haplotype carriers were expected (P = 0.018). Whole genome-sequencing uncovered that both asthenospermic bulls were homozygous for a mutation that disrupts a canonical 5' splice donor site of CCDC189 encoding the coiled-coil domain containing protein 189. Transcription analysis showed that the derived allele activates a cryptic splice site resulting in a frameshift and premature termination of translation. The mutated CCDC189 protein is truncated by more than 40%, thus lacking the flagellar C1a complex subunit C1a-32 that is supposed to modulate the physiological movement of the sperm flagella. The mutant allele occurs at a frequency of 2.5% in Nordic Red cattle.
Our study in cattle uncovered that CCDC189 is required for physiological movement of sperm flagella thus enabling active progression of spermatozoa and fertilization. A direct gene test may be implemented to monitor the asthenospermia-associated allele and prevent the birth of homozygous bulls that are infertile. Our results have been integrated in the Online Mendelian Inheritance in Animals (OMIA) database ( https://omia.org/OMIA002167/9913/ ).
Ciliopathies presenting as inherited hepatorenal fibrocystic disorders are rare in humans and in dogs. We describe here a novel lethal ciliopathy in Norwich Terrier puppies that was diagnosed at ...necropsy and characterized as diffuse cystic renal disease and hepatic fibrosis. The histopathological findings were typical for cystic renal dysplasia in which the cysts were located in the straight portion of the proximal tubule, and thin descending and ascending limbs of Henle's loop. The pedigree of the affected puppies was suggestive of an autosomal recessive inheritance and therefore, whole exome sequencing and homozygosity mapping were used for identification of the causative variant. The analyses revealed a case-specific homozygous splice donor site variant in a cilia related gene, INPP5E: c.1572+5G>A. Association of the variant with the defect was validated in a large cohort of Norwich Terriers with 3 cases and 480 controls, the carrier frequency being 6%. We observed that the identified variant introduces a novel splice site in INPP5E causing a frameshift and formation of a premature stop codon. In conclusion, our results suggest that the INPP5E: c.1572+5G>A variant is causal for the ciliopathy in Norwich Terriers. Therefore, genetic testing can be carried out in the future for the eradication of the disease from the breed.
Male infertility is an increasing problem in all domestic species including man. Localization and identification of genes involved in defects causing male infertility provide valuable information of ...specific events in sperm development. Sperm development is a complex process, where diploid spermatogonia develop into haploid, highly specialized spermatozoa. Correct expression and function of various genes and their protein products are required for production of fertile sperm. We have identified an infertility defect in Finnish Yorkshire boars caused by spermatogenic arrest. The aim of this study was to locate the disease associated region using genome wide screen with the PorcineSNP60 Beadchip and identify the causal mutation by candidate gene approach.
In the Finnish Yorkshire pig population the spermatogenic arrest (SA) defect appears to be of genetic origin and causes severe degeneration of germ cells and total absence of spermatozoa. Genome wide scan with the PorcineSNP60 Beadchip localized the SA defect to porcine chromosome 12 in a 2 Mbp region. Sequencing of a candidate gene Tex14 revealed a 51 bp insertion within exon 27, which caused differential splicing of the exon and created a premature translation stop codon. The expression of Tex14 was markedly down regulated in the testis of a SA affected boar compared to control boars and no protein product was identified by Western blotting. The SA insertion sequence was also found within intron 27 in all analyzed animals, thus the insertion appears to be a possible duplication event.
In this study we report the identification of a causal mutation for infertility caused by spermatogenic arrest at an early meiotic phase. Our results highlight the role of TEX14 specifically in spermatogenesis and the importance of specific genomic remodeling events as causes for inherited defects.
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