Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Comparison of mammalian SPANX-N proteins. (a) Alignment of the conserved core between human SPANX-N proteins and homologs in non-primate mammals. With the exception of rat SPANX-N, the core starts immediately at the N-terminus. The plot highlights physiochemical properties, and the sequence conservation is shown as a consensus at the bottom. (b) Polymorphic positions in the human and dog coding sequences (only core shown). Two nonsynonymous changes, N21D and S79N (not shown), were found in the four dog and two wolf alleles analyzed. The five compared SPANX-N2 alleles revealed a nonsynonymous change, T8I, and a synonymous polymorphism in codons 4, 80, and 151. One amino acid replacement, K43N, was found in the two compared SPANX-N3 alleles. The two analyzed SPANX-N4 alleles revealed only one nonsynonymous change, K48N. (c) Substitutions in chimpanzee SPANX-N2 and -N3 coding sequences compared to human homologs. Chimpanzee SPANX-N2 contains four nonsynomous substitutions, two of which are in the core (K43N and Y55H). In addition, there are three synonymous changes and a 65 aa long deletion caused by the deletion of five 39 bp minisatellite units. Chimpanzee SPANX-N3 contains 10 nonsynonymous changes compared to human SPANX-N3, with three in the conserved core (E18K, N21S, and K23E). There is also a single aa deletion, del22K, and two synonymous changes. (d) Phylogenetic relationship of SPANX-N proteins in mammals obtained using the maximum likelihood method. (e) Minisatellite variations in the C-terminal part of primate SPANX-N genes. With the exception of the human SPANX-N4 locus, the C-terminal regions contain 39 bp minisatellite arrays (blue). In some cases, the translation termination codon (red) is located after the array, and the repeats encode the C-terminal portion of SPANX-N genes.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Evolution of SPANX-N genes. The probable scheme of evolution of the SPANX-N gene family is based on the pairwise alignments and breakpoint analysis. The exons are marked red in colour, intron regions are marked in pink. Positions relative to the centromere (cen) and telomere (qter) are shown. The numbers near duplication breakpoints indicate positions of the SPANX-N regions in the human genome (hg16; UCSC March 2006 genome version). The most likely scenario is that SPANX-N3 was the original locus.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques:

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: An evolutionary history of the SPANX gene family. The common ancestor of rodents, canine, and primates apparently had a single SPANX-N subfamily gene. An extensive search for potential divergent members of the SPANX-N family in the mouse, rat, dog and wolf genomes failed to detect any. A single SPANX gene is marked as a grey box. Orangutan and rhesus macaque have three and four copies of SPANX-N genes, correspondingly (boxes in blue). The emergence of the SPANX-A / D gene subfamily (boxes in red) is a more recent event, subsequent to the separation of the hominoid lineage from orangutan and rhesus macaque. Apparently, this subfamily evolved via duplication of one of the SPANX-N genes accompanied by deletion of the distal part of exon 2 (minisatellites) and rapid divergence. SPANX-A/D genes are impeded in segmental duplications (boxes in yellow). African Great Apes (bonobo, chimpanzee and gorilla) have four members of the SPANX-A/D subfamily. Notably, duplication of SPANX-C and amplification of SPANX-B genes from 1 to 14 copies appears to be human lineage specific.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Amplification

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Indirect immunofluorescent staining of fixed, permeabilized swim-up spermatozoa with antibodies against SPANX-N proteins (in green). The SPANX-N immunofluorescence is observed in the acrosome of spermatozoa. The SPANX-A/D immunofluorescence is observed in association with nuclear craters in the cytoplasmic droplet at the posterior sperm head, or in both (red dots). DNA staining with DAPI is in blue.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Immunostaining of SPANX-N (a, b) and the negative control (c, d) at low (a, c) and high magnification (b, d) on normal human testis sections (bars indicated). The staining is clearly specific for late spermatids and spermatozoa (in pink, a, b). Autofluorescence (i.e., non-specific signal, a, b) is also detectable in red blood cells and can be recognized by the overlapping signals in the green and red wavelengths. Nuclei are counterstained with DAPI (in blue).

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Immunostaining, Negative Control, Staining

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: (a) RT-PCR analysis of the SPANX-N gene subfamily in normal adults tissues and cancer cell lines. cDNA was prepared from a panel of human tissue mRNAs and cell lines. Oligonucleotides were designed within exons 1 and 2 to amplify putative transcripts. The observed bands of the expected size 180 bp were sequenced and confirmed to correspond to SPANX-N genes. The strongest expression of SPANX-N genes was observed in the normal testis (lanes 12 and 17) and in the LOXIMV1 melanoma cell line (lanes 13 and 18). Lanes 2–11 correspond to normal and tumor pairs of breast, cervix, prostate, lung and ovary; lanes 19–21 correspond to 938MEL, 888MEL, SKMEL28 melanoma cell lines; lane 22 corresponds to the SKOV3 ovarian cell line; lanes 1, 15, 16, 24 – ladder; lanes 14 and 23 - water. The cDNA templates used were normalized using actin, as shown at the bottom of the panel. (b) Western blot analysis of lysates from normal tissues using an anti-EQPT antibody. Lane 1 - lung, lane 2 - testis, lane 3 - placenta and lane 4 – prostate. Lanes 5–9: full-size SPANX-N proteins expressed in the pET-11d in Bl21 cells. The mobility of SPANX-N proteins produced in E. coli cells is 13 kDa for SPANX-N1, 27 kDa for SPANX-N2, 23 kDa for SPANX-N3, 17 kDa for SPANX-N4 and 13 kDa for SPANX-N5.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Produced

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Immunocytochemical detection of SPANX-N in the melanoma cell line. 938MEL melanoma cell line (green; middle and right panels); co-immunostaining for the cell multiplication marker Ki67 (red; left and right panels; overlap with SPANX-N appears as yellow). Upper panels show the test samples, whereas bottom panels provide the negative control for SPANX-N (primary antibody omitted).

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Immunostaining, Marker, Negative Control

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: Comparison of human SPANX-A/D and SPANX-N promoters. Detected transcription starts are marked in yellow, the translation initiation codons ATG are marked in green. Noncoding sequences are in lowercase. SPANX-N copies differ from SPANX-A/D genes by the almost complete lack of all CpG dinucleotides in the promoter regions (in orange); however, these CpGs are perfectly preserved in all of the SPANX-A/D copies. CpG sites are marked only on one strand. Sp1 binding consensus in four SPANX-N copies is marked in blue. CTCF-binding sites are marked by red boxes.

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

doi: 10.1371/journal.pone.0000359

Figure Lengend Snippet: In vitro interaction of CTCF with the promoter sequences of SPANX genes. (a) EMSA was carried out with either control lysate (−) or lysate containing the in vitro translated 11 ZF DNA binding domain of CTCF protein (+). The positions of the bound CTCF-DNA complexes, containing the 11ZF domain, are indicated on the left by arrow (shift). Free DNA probe is also indicated (free). Control: a positive control (c-myc promoter) of the EMSA reaction. (b) A schematic representation of the overlapping fragments of the SPANX-N promoter DNA sequences used in EMSA. The −195 to −43 bp is the smallest fragment showing retarded migration (red box). The ATG is considered as +1. On the right is CTCF binding of the fragments (+ positive; − negative).

Article Snippet: Notably, mobility of SPANX-N proteins is approximately 6 kDa higher than that predicted from their coding regions (8 kDa, 20 kDa, 16 kDa, 11 kDa and 8 kDa for SPANX-N1, -N2, -N3, -N4 and –N5, correspondingly).

Techniques: In Vitro, Binding Assay, Positive Control, Migration

Journal: The EPMA Journal

Article Title: Implementing a preimplantation proteomic approach to advance assisted reproduction technologies in the framework of predictive, preventive, and personalized medicine

doi: 10.1007/s13167-022-00282-5

Figure Lengend Snippet: Potential biomarkers of male infertility

Article Snippet: , AKAP4 AKAP4pre, ODFP, SPANX PSMA1, PIPpre , Structural, binding, sperm capacitation , Down , [ ] .

Techniques: Binding Assay, Membrane, Ubiquitin Proteomics, Activity Assay, Protein Binding, Inhibition, Cell Differentiation, Expressing, Agglutination, Lysis