ATR FT-IR (1100–1800 cm⁻¹) spectra obtained from thin hydrated-films containing mature amyloid-like fibrils.

<p>These thin hydrated-films, formed from (a) ZPH_A, (b) ZPH_G and (c) the mixture of ZPH_A & ZPH_G peptides, were cast on flat stainless-steel plates coated with an ultra thin hydrophobic layer (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’). Second derivative spectra are also included and were used for the exact identification of the band maxima and their tentative assignments. All resulting spectra are indicative of the preponderance of an antiparallel β-sheet secondary structure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t002" target="_blank">Table 2</a>).</p

Bands observed in the IR spectrum of a hydrated film produced from a suspension of fibrils produced by ZPH_A peptide, ZPH_G peptide and from a mixture of both peptides, dissolved in equal (1∶1) amounts and their tentative assignments (Fig. 5).

<p>Bands observed in the IR spectrum of a hydrated film produced from a suspension of fibrils produced by ZPH_A peptide, ZPH_G peptide and from a mixture of both peptides, dissolved in equal (1∶1) amounts and their tentative assignments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-g005" target="_blank">Fig. 5</a>).</p

Photomicrographs of peptide fibrils stained with Congo red.

<p>Fibrils have derived from: ZPH_A (a–b), ZPH_G (c–d) and ZPH_A & ZPH_G mixture (e–f) peptides, respectively (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’). Solutions of these peptides, after ca. one (1) week incubation, produced fibrils, which were then stained with Congo red. The typical for amyloid fibrils apple-green birefringence is clearly seen, under crossed polars. (a,c,e) Bright field illumination, (b,d,f) Crossed polars Bar 400 µm.</p

Sequence alignment of the ZP-N domain of mouse ZP3 and human ZP1 proteins.

<p>The crystallographically determined secondary structure elements are depicted below the sequences: Arrows and helices represent observed beta-strands (named consecutively A to G) and alpha helices, respectively. The invariant cysteine residues connected by disulfide bonds (dotted lines) are also seen. Peptides ATVQCF and FQLHVRC, corresponding to the beta strands A and G of human ZP1, respectively, predicted by AMYLPRED <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Frousios1" target="_blank">[43]</a> as ‘aggregation-prone’ stretches, are enclosed in boxes (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’).</p

Electron micrographs of amyloid-like fibrils, negatively stained with 1% uranyl acetate.

<p>Amyloid-like fibrils were derived by self-assembly, from a 10 mg ml⁻¹ solution of peptides ZPH_A (a) and ZPH_G (b) in distilled water, pH 5.5. A solution of a mixture of the ZPH_A and ZPH_G peptides (in 1∶1 ratio, in distilled water, pH 5.5, concentration 5 mg ml⁻¹ per peptide) also revealed the formation of amyloid-like fibrils, after an incubation period of ca. one week (c). (a) They are unbranched and of undetermined length, approximately 100–120 Å in diameter and have a double helical structure. A pair of protofilaments each 40–50 Å in diameter wrap around each other, with intervening stain between them, thus forming double-helical fibrils (arrows). Bar 500 nm. (b) Protofilaments interact laterally, forming ribbons and, eventually, gels. The fibrils formed exhibit a characteristic for amyloid-like fibrils polymorphism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Kodali1" target="_blank">[67]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Pedersen1" target="_blank">[69]</a>. Bar 200 nm. (c) Two different types (populations) of fibrils are apparent, due to “self-aggregation” of each peptide (double and single arrows, respectively), which are similar to those viewed separately by the ZPH_A and ZPH_G peptide solutions in (a) and (b) above. Bar 500 nm.</p

X-ray diffraction patterns produced from oriented fibres of mature fibril suspensions.

<p>The mature fibrils have derived from: (a) ZPH_A peptide, (b) ZPH_G peptide, (c) a mixture of ZPH_A & ZPH_G peptides. The meridian, M (direction parallel to the fibre axis, F) is vertical and the equator, E, is horizontal in this display. All X-ray diffraction patterns are clearly “cross-β” patterns <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Geddes1" target="_blank">[71]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Jahn1" target="_blank">[73]</a>. (a) An intense meridional 4.7 Å reflection corresponds to the spacing of successive hydrogen bonded β-strands, perpendicular to the fiber axis, whereas the 9.1 Å reflection on the equator is attributed to the packing distance of β-sheets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 4). The sheets are packed parallel to the fiber axis. (b) The X-ray diffraction pattern of the ZPH_G peptide also exhibits similar reflections that indicate the presence of a “cross-β” conformation. The structural repeat of 4.7 Å corresponds to the spacing of successive β-strands arranged perpendicular to the fiber axis, while the 12.4 Å spacing on the equator, corresponds to the packing distance of consecutive β-sheet parallel to the fibre axis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 5). (c) The X-ray pattern produced from the mixture of the ZPH_A & ZPH_G peptide fibril suspensions is clearly a combination of the diffraction patterns produced by the individual fibers formed from the ZPH_A and ZPH_G peptide's fibril suspensions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 6). The 2.4 Å, 3.8 Å, 7.1 Å and 9.1 Å reflections are due to the presence of fibrils formed by the ZPH_A peptide, whereas the 12.4 Å reflection is produced by fibrils formed by the ZPH_G peptide (corresponding to β-sheet packing distance). The intense 4.7 Å reflection has contributions from both fibril populations and this is in agreement with the EM photograph of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-g002" target="_blank">Fig. 2c</a>.</p

Improving ICSI: A review from the spermatozoon perspective

<p>Intracytoplasmic sperm injection (ICSI) is the most frequently applied method for fertilization making the process of identifying the perfect spermatozoon fundamental. Herein we offer a critical and thorough presentation on the techniques reported regarding (i) handling and preparing semen samples, (ii) identifying and ‘fishing’ spermatozoa, and (iii) improving key factors, such as motility for a successful ICSI practice. These approaches are suggested to make the process easier and more effective especially in atypical and challenging circumstances. Furthermore, we present an epigrammatic opinion-where appropriate-based upon our collective experience. Techniques such as intracytoplasmic morphologically selected sperm injection, hyaluronic binding, polarized light microscopy, and annexin V agent identification for comparing sperm cells and their chromatin integrity are analyzed. Moreover, for the demanding cases of total sperm immotility the use of the hypoosmotic swelling test, methylxanthines, as well as the option of laser assisted immotile sperm selection are discussed. Finally, we refer to the employment of myoinositol as a way to bioreactively improve ICSI outcome for oligoasthenoteratozoospermic men. The diversity and the constant development of novel promising techniques to improve ICSI from the spermatozoon perspective, is certainly worth pursuing. The majority of the techniques discussed are still a long way from being established in routine practices of the standard IVF laboratory. In most cases an experienced embryologist could yield the same results. Although some of the techniques show great benefits, there is a need for large scale multicenter randomized control studies to be conducted in order to specify their importance before suggesting horizontal application. Taking into consideration the <i>a priori</i> invasive nature of ICSI, when clinical application becomes a possibility we need to proceed with caution and ensure that in the pursuit for innovation we are not sacrificing safety and the balance of the physiological and biological pathways of the spermatozoon’s dynamic.</p> <p><b>Abbreviations:</b> ICSI: intracytoplasmic sperm injection; IVF: <i>in vitro</i> fertilization; PGD: reimplantation genetic diagnosis; IVM: <i>in vitro</i> maturation; HCV/HIV: hepatitis C virus/human immunodeficiency virus; IMSI: intracytoplasmic morphologically selected sperm injection; DGC: density gradient centrifugations; S-U: swim-up; ART: assisted reproduction technology; IUI: intrauterine insemination; PVP: polyvinylpyrrolidone; HA: hyaluronic acid; MSOME: motile sperm organelle morphology examination; ZP: zona pellucida; MACS: magnetic activation cell sorting; HOST: hypo-osmotic swelling test; TESE: testicular sperm extraction; MMP: mitochondrial membrane potential; OAT: oligoasthenoteratozoospermic</p