Anu Sironen

Transcriptome Profiling of the Murine Testis during the First Wave of Spermatogenesis

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.

Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia

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.

KIF3A is essential for sperm tail formation and manchette function.

KIF3A motor protein is responsible for intraflagellar transport, which is required for protein delivery during axoneme formation in ciliated cells. The function of KIF3A during spermatogenesis is not known. In this study, we show that depletion of KIF3A causes severe impairments in sperm tail formation and interestingly, it also affects manchette organization and the shaping of sperm heads. Our results demonstrate the analogy between the mechanisms governing the formation of cilia in somatic cells and the formation of spermatozoa-specific flagella. Furthermore, this study reveals KIF3A as an important regulator of spermatogenesis and emphasizes the crucial role of KIF3A in maintaining male fertility. We also identified several novel interacting partners for KIF3A, including meiosis-specific nuclear structural protein 1 (MNS1) that colocalizes with KIF3A in the manchette and principal piece of the sperm tail. This study highlights the essential role of KIF3A-mediated microtubular transport in the development of spermatozoa and male fertility.

Expression of SPEF2 During Mouse Spermatogenesis and Identification of IFT20 as an Interacting Protein

Abstract 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.

Formation and function of the manchette and flagellum during spermatogenesis.

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 intra-flagellar 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.

Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed

BackgroundGood genetic progress for pig reproduction traits has been achieved using a quantitative genetics-based multi-trait BLUP evaluation system. At present, whole-genome single nucleotide polymorphisms (SNP) panels provide a new tool for pig selection. The purpose of this study was to identify SNP associated with reproduction traits in the Finnish Landrace pig breed using the Illumina PorcineSNP60 BeadChip.MethodsAssociation of each SNP with different traits was tested with a weighted linear model, using SNP genotype as a covariate and animal as a random variable. Deregressed estimated breeding values of the progeny tested boars were used as the dependent variable and weights were based on their reliabilities. Statistical significance of the associations was based on Bonferroni-corrected P-values.ResultsDeregressed estimated breeding values were available for 328 genotyped boars. Of the 62 163 SNP in the chip, 57 868 SNP had a call rate > 0.9 and 7 632 SNP were monomorphic. Statistically significant results (P-value < 2.0E-06) were obtained for total number of piglets born in first and later parities and piglet mortality between birth and weaning in later parity, and suggestive associations (P-value < 4.0E-06) for piglet mortality between birth and weaning in first parity, number of stillborn piglets in later parity, first farrowing interval and second farrowing interval. Two of the statistically significant regions for total number of piglets born in first and later parities are located on chromosome 9 around 95 and 79 Mb. The estimated SNP effect in these regions was approximately one piglet between the two homozygote classes. By combining the two most significant SNP in these regions, favourable double homozygote animals are expected to have 1.3 piglets (P-value = 1.69E-08) more than unfavourable double homozygote animals. A region on chromosome 9 (66 Mb) was statistically significant for piglet mortality between birth and weaning in later parity (0.44 piglets between homozygotes, P-value = 6.94E-08).ConclusionsThree separate regions on chromosome 9 gave significant results for litter size and pig mortality. The frequencies of favourable alleles of the significant SNP are moderate in the Finnish Landrace population and these SNP are thus valuable candidates for possible marker-assisted selection.

Mapping of an immotile short tail sperm defect in the Finnish Yorkshire on porcine Chromosome 16

An immotile short tail sperm defect has recently been identified as a hereditary disorder present within the Finnish Yorkshire pig population. The syndrome is inherited as an autosomal recessive disease exclusively expressed in male individuals as shorter sperm tail length and immotile spermatozoa. Based on the assumption of a recent common origin of the disease-causing mutation, a genome-wide search was performed with 228 evenly spaced microsatellites by homozygosity mapping of affected and unaffected DNA pools. One locus, SW2411 on Chr 16, demonstrated a significantly skewed allele distribution between the two pools. Linkage analysis of five markers in this region mapped the disease-causing gene within a 6-cM confidence interval region with a highest LOD score of 7.7 at marker SW419. It appears that three-marker haplotypes can be used for marker-assisted selection within analyzed pedigrees. Furthermore, future fine mapping may reveal a more precise population-wide associated haplotype and facilitate identification of a new gene affecting sperm tail development.

Effect of polymorphisms in candidate genes on reproduction traits in Finnish pig populations.

Reproduction traits play an important role in economically viable piglet production and are closely related to the quality and length of the productive life of the sow. A increased removal rate of young sows is undesirable not only because of the associated financial penalties incurred, but also because of ethical concerns. Candidate genes and gene pathways have been identified for fertility in model species, and recent studies have provided evidence that polymorphisms within these genes are associated with reproduction traits in American Yorkshire/Large White and Landrace populations. In this study we evaluated the impact of single polymorphisms (n = 7) in 7 candidate genes on reproductive efficiency in Finnish Yorkshire (n = 280) and Landrace (n = 271) populations: IGFBP1, IGFBP2, IGFBP3, IGFBP5, CPTIA (carnitine O-palmitoyltransferase I), COX2 (PG-endoperoxide synthase 2, also known as cyclooxygenase-2), and SLC22A5 [organic cation/carnitine transporter 2 (solute carrier family member I), OCTN2]. In the Finnish Yorkshire population, only 4 of the analyzed markers were polymorphic. Significant effects on farrowing time were detected from the Yorkshire data, with polymorphisms within the genes CPT1A [a (allele substitution effect of allele A) = 2.97 d for age at first farrowing)], IGFBP3 (a = 0.54 d for farrowing interval of parities >1), and IGFBP5 (a = 3.22, 1.27, and 0.85 d for age at first farrowing and farrowing interval in the first and later parities, respectively). For the Landrace population, 6 markers were polymorphic, and significant effects were detected for traits affecting litter size. The polymorphism within the COX2 gene had an additive effect of 0.3 piglets for litter size in parities >1, and the IGFBP1 gene had an additive effect of 0.21, 0.26, and 0.11 piglets for litter size in the first parity, parities >1, and stillborn in parities >1, respectively. The additive effect of the SNP within the IGFBP2 gene was 0.16, 0.09, and 0.09 piglets for litter size in parities >1 and stillborn in the first and later parities, respectively. Finally, the IGFBP5 gene had an additive effect of 0.18, 0.07, and 0.07 piglets for litter size in the first parity, stillborn in parities >1, and mortality between farrowing and weaning in the first parity, respectively. These results support the suitability of the candidate gene approach for identification of markers to improve the reproductive performance of sows and to provide potential markers for marker-assisted selection.

Knobbed acrosome defect is associated with a region containing the genes STK17b and HECW2 on porcine chromosome 15

BackgroundMale 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. Correct condensation of the sperm head and development of the acrosome are required for fertile sperm. In the Finnish Yorkshire pig population a knobbed acrosome defect (KAD) has been reported which appears to be of genetic origin. In previous studies we have shown that a large number of affected spermatozoa have a cystic swelling anterior to the apical part of the acrosome.ResultsCharacterization of the knobbed acrosome affected sperm revealed that both the acrosomal granules and chromatin are affected. This type of KAD appears to be a previously unknown and serious form of the defect. A genome wide scan with PorcineSNP60 Genotyping BeadChip defined the KAD associated region within 0.7 Mbp on porcine chromosome 15. Two genes, STK17b and HECW2, located within this region were sequenced. The expression of these genes appeared comparable in KA-affected and control boars. The known function of HECW2 in acrosome development highlighted this gene as a good candidate responsible for the KAD. One nonsynonymous SNP was identified within the HECW2 gene. However, as this mutation was found in homozygous state in individuals with normal sperm, this is not likely to be the causal mutation.ConclusionsIn this study we identified two candidate genes for a severe defect affecting both the sperm acrosome and chromatin that causes infertility. One of these genes, HECW2, plays an important role in ubiquitination, a prerequisite for chromatin remodelling and acrosome formation, highlighting the involvement of this gene in the knobbed acrosome defect and male infertility.

Evidence for three highly significant QTL for meat quality traits in the Finnish Yorkshire pig breed

Meat quality is important both to consumers and to the meat processing industry. Commonly used measures of porcine meat quality are the pH and color of the meat. The purpose of this study was to identify SNP associated with these meat quality traits in Finnish Yorkshire using the Illumina PorcineSNP60 BeadChip. The association of each SNP with the quality traits was tested with a weighted linear model. The relatedness of samples was accounted for by a random polygenic genetic effect with the accompanying full relationship matrix. The original EBV from single-trait evaluations were deregressed before analysis. The statistical significance of SNP was established using the Bonferroni correction to adjust for multiple testing. Three genomic regions were significant for the meat quality traits. The PRKAG3 region on chromosome 15 was significant for pH measured from loin and ham and for a* (redness) measured from loin. The smallest P-value in the region was obtained for pH measured from loin (ASGA0070634, P-value = 3.8 × 10(-13)). The allele substitution effect (-0.047) of the unfavorable allele A corresponds to 1 SD of the polygenic effect. The second significant region, on chromosome 2 at around 31 megabases (Mb), was associated with pH and L* (lightness) measured from loin. The most significant SNP (ASGA0009814, P-value = 3.89 × 10(-10)) had an allele substitution effect of 0.86, corresponding to 0.7 SD of the polygenic effect of L*. The third region, located on chromosome 6 at around 83 Mb, was significant for a* measured from ham. The P-value of the best SNP (ALGA0035896) was 8.71 × 10(-7) and the allele substitution effect -0.38, corresponding to 0.5 SD of the polygenic effect of a*. The significant association of PRKAG3 with pH was not due to the known AA substitutions. The candidate gene on chromosome 2 associated with color L* is RCN1, which has a high affinity Ca(2+)-binding motif, the EF hand. The significant region on chromosome 6 for color a* contains several genes, so more data are needed to identify the causative gene. Our results indicate that instead of the known AA substitutions of PRKAG3, some yet-unknown AA substitutions are causative for the pH variation in Finnish Yorkshire. Also, a new major QLT for L* was found on chromosome 2. The significant SNP identified in this study can be used in marker-assisted selection.