Sperm Flagellar Protein Research Articles

Integrative omics reveals clues as to why Cynoglossus semilaevis pseudomales produce no W sperm

Cynoglossus semilaevis (half-smooth tongue sole) is an important aquaculture species that has a ZW/ZZ sex-determination system (females, ZW; males, ZZ). Some genotypic females may develop phenotypic males, and are known as pseudomales (ZW). These pseudomales can develop mature testes and produce sperm. However, previous studies have shown that pseudomales cannot produce sperms bearing W chromosome (W sperm). In this study, transcriptomic, proteomic, and ubiquitinated proteomic analyses were employed simultaneously to investigate the differences in gonads between pseudomales and normal males. Functional enrichment analysis of the differentially expressed genes/proteins showed that non-coding RNA metabolic processes and RNA degradation were more active in the gonads of pseudomales, and they showed higher levels of ubiquitination compared to males. In addition, most genes/proteins related to sperm morphogenesis expressed downwards in pseudomales. These genes included genes located exclusively on the Z chromosome, such as sperm flagellar protein 2 (spef2), doublesex and mab-3 related transcription factor 1 (dmrt1), doublesex and mab-3 related transcription factor 3 (dmrt3), and cilia- and flagella-associated protein 157 (cfap157). These findings characterized the transcriptional and proteomic profiles of the gonads of pseudomales and males, and suggested that W sperm were absent in pseudomales, possibly due to the disruption of sperm morphogenesis, resulting from W chromosome lacking spermatogenesis-related genes.

Cilia-related protein SPEF2 regulates osteoblast differentiation

Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.

Identification of candidate genes for chicken early- and late-feathering

Previous studies suggest that prolactin receptor (Prlr) is a potential causative gene for chicken early- (EF) and late-feathering (LF) phenotypes. In this study, we evaluated candidate genes for this trait and determined the expression of 3 genes, including Prlr, sperm flagellar protein 2 (Spef2), and their fusion gene, in the skins of one-day-old EF and LF chicks using RT­qPCR. Data indicated that Prlr expression in the skin did not show significant difference between EF and LF chicks, suggesting Prlr may not be a suitable candidate gene. In contrast, Spef2 expression in the skin displayed a significant difference between EF and LF chicks (P < 0.01), suggesting that Spef2 may be a good candidate gene for chicken feathering. Moreover, dPrlr/dSpef2, the fusion gene, was also a good candidate gene as it was expressed only in LF chicks. However, the expression of the fusion gene was much lower than that of Prlr. Additionally, using strand-specific primers, we found that the fusion gene was transcribed in 2 directions (one from dPrlr promoter, another from dSpef2 promoter), which could result in the formation of a double strand RNA. In conclusion, both Spef2 and the fusion gene are good candidate genes for chicken feathering, but Prlr is not. The research on the function and regulation of the candidate genes will help elucidate the molecular basis of the chicken feathering trait.

Describing the potential crossreactome between mumps virus and spermatogenesis-associated proteins.

This study documents that mumps virus shares several peptide sequences with human proteins that, when altered, may be involved in oligospermia/azoospermia, sterility and testicular atrophy. Examples are sperm flagellar protein 2 and spermatogenesis and centriole-associated protein 1. The data invite to further investigate crossreactivity as a potential mechanism linking anti-mumps immune responses, alterations of spermatogenesis-associated antigens and male fertility disorders.

A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus.

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1

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.

Evaluation of Contraceptive Potential of a Novel Epididymal Sperm Protein SFP2 in a Mouse Model

Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice. Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization. Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility. These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target.

Involvement of redox- and phosphorylation-dependent pathways in osmotic adaptation in sperm cells of euryhaline tilapia

Sperm cells involved in fertilisation must tolerate hypo-osmotic and hyper-osmotic environments. Euryhaline tilapia (Oreochromis mossambicus) can acclimatise to and reproduce in freshwater and seawater because its sperm are able to adapt to these differing osmotic environments. In this study, we found that the dephosphorylation of sperm proteins in O. mossambicus correlated with the activation of flagellar motility when sperm were exposed to hypotonic or hypertonic conditions, and that differences in phosphorylation may reflect adaptations to a given osmotic environment. Of the sperm proteins that were dephosphorylated, the phosphorylation pattern of an 18 kDa protein, identified as the superoxide anion scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), was different in freshwater- and seawater-acclimatised tilapia sperm. Cu/Zn SOD was distributed from the sperm head to the flagellum. Additionally, differences were observed between freshwater and seawater tilapia in the nitration of tyrosine residues (which might be mediated by SOD) in sperm flagellar proteins in response to osmotic shock. These results demonstrate that reactive-oxygen-species-dependent mechanisms contribute to both osmotic tolerance and the activation of flagellar motility.

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Partial duplication of the PRLR and SPEF2 genes at the late feathering locus in chicken

BackgroundOne of the loci responsible for feather development in chickens is K. The K allele is partially dominant to the k+ allele and causes a retard in the emergence of flight feathers at hatch. The K locus is sex linked and located on the Z chromosome. Therefore, the locus can be utilized to produce phenotypes that identify the sexes of chicks at hatch. Previous studies on the organization of the K allele concluded the integration of endogenous retrovirus 21 (ev21) into one of two large homologous segments located on the Z chromosome of late feathering chickens. In this study, a detailed molecular analysis of the K locus and a DNA test to distinguish between homozygous and heterozygous late feathering males are presented.ResultsThe K locus was investigated with quantitative PCR by examining copy number variations in a total of fourteen markers surrounding the ev21 integration site. The results showed a duplication at the K allele and sequence analysis of the breakpoint junction indicated a tandem duplication of 176,324 basepairs. The tandem duplication of this region results in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Sequence analysis revealed that the duplication is similar in Broiler and White Leghorn. In addition, twelve late feathering animals, including Broiler, White Leghorn, and Brown Layer lines, contained a 78 bp breakpoint junction fragment, indicating that the duplication is similar in all breeds. The breakpoint junction was used to develop a TaqMan-based quantitative PCR test to allow distinction between homozygous and heterozygous late feathering males. In total, 85.3% of the animals tested were correctly assigned, 14.7% were unassigned and no animals were incorrectly assigned.ConclusionThe detailed molecular analysis presented in this study revealed the presence of a tandem duplication in the K allele. The duplication resulted in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Furthermore, a DNA test was developed to distinguish between homozygous and heterozygous late feathering males.

Preferential Localization of Rat GAPDS on the Ribs of Fibrous Sheath of Sperm Flagellum and Its Expression during Flagellar Formation

The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium

The olfactory epithelium has the unusual ability to replace its neurons. We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5–7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlh1, Hes6, Lmyc1, c-Myc, Mxd4, Id1, Nmyc1, Cited2, c-Myb, Mybl1, Tead2, Dp1, Gata2, Lmo1, and Sox11. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

The expression and cellular localization of the sperm flagellar protein MC31/CE9 in the rat testis: possible posttranscriptional regulation during rat spermiogenesis.

We isolated the MC31 cDNA clone coding the antigen specifically recognized by the monoclonal antibody mMC31, and found that MC31 was identical to rat CE9. Therefore, this molecule is called MC31/CE9. MC31/CE9, a member of the immunoglobulin superfamily molecules, was localized on the rat sperm flagellar plasma membrane. We analyzed the expression and cellular localization of MC31/CE9 mRNA and protein in the adult rat testis by use of Northern hybridization, in situ hybridization, and immunohistochemical analyses. In the course of spermatogenesis, MC31/CE9 mRNA first appeared in type B spermatogonia. The mRNA signal intensity increased progressively to pachytene spermatocytes and remained constantly at a considerable level throughout the subsequent phases of spermatocytes and round spermatids, and then decreased gradually from step-11 spermatids to disappear in step-15 spermatids. On the other hand, MC31/CE9 protein expression showed a bimodal pattern. Immunohistochemical analysis for the MC31/CE9 protein revealed its most intense immunoreactivity on the flagella of step-8 to step-19 elongated spermatids. The cytoplasmic immunoreactivity of the MC31/CE9 protein also appeared in preleptotene to early pachytene spermatocytes and elongated spermatids, with particularly intense immunoreactivity in the Golgi complexes of zygotene and early pachytene spermatocytes (stage XIII to III) as well as step-8 to step-13 spermatids. Between these two phases, the MC31/CE9 protein proved undetectable in the cytoplasm of any spermatogenic cells. Sertoli cells and Leydig cells were devoid of MC31/CE9 mRNA and its protein. Therefore, the production of MC31/CE9 is thought to be posttranscriptionally regulated during spermiogenesis.

Phosphorylation of axonemal 21 kDa and 26 kDa proteins modulates activation of sperm motility in the ascidian,Ciona intestinalis

Protein phosphorylation is highly coupled with sperm motility activation in several animal species. The micro-tubule based flagellar motor protein, dynein, is a candidate for a phosphoprotein related to sperm activation in many animal species (Morisawa &amp; Hayashi, 1985; Hayashiet al., 1987; Dey &amp; Brokaw, 1991; Stephens &amp; Prior, 1992; Inabaet al., 1998, 1999). Sperm motility of the ascidiansCiona intestinalisandC. savignyiis activated by a factor derived from unfertilised eggs named sperm activating and attracting factor (SAAF). SAAF elevates the intracellular cyclic AMP (cAMP) level by a mechanism dependent on membrane hyperpolarisation and extracellular Ca2+(Yoshidaet al., 1994; Izumiet al., 1999). Experiments using demembranatedCionasperm showed that cAMP is required prior to ATP for the activation of axonemal movement (Opreska &amp; Brokaw, 1983; Morisawaet al., 1984; Brokaw, 1985; Dey &amp; Brokaw, 1991; Chaudhryet al., 1995) and that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (Dey &amp; Brokaw, 1991). However, there is no evidence of which proteins are phosphorylated during the SAAF-dependent activation ofCionasperm motility.