Glass transition temperatures of solutions composed of different contents of DMSO and sucrose.

<p>The onset temperature of glass transition was determined using DSC. This was done for 0–20% (v/v) DMSO solutions without supplements (white circles), as well as supplemented with 0.5 M sucrose (grey circles) or 1 M sucrose (black circles). For liposome storage experiments, performed at −80°C (dotted line), four different formulations were selected (labeled S1–4). Solution S1 was composed of 5% DMSO with 1 M sucrose (T_g: −68°C), S2 of 10% DMSO with 1 M sucrose (T_g: −77°C), S3 of 5% DMSO with 0.5 M sucrose (T_g: −84°C) and S4 of 10% DMSO with 0.5 M sucrose (T_g: −101°C).</p

Water fraction distributions in solutions composed of DMSO and sucrose, determined from the OH-stretching band in FTIR spectra.

<p>In panel A–B, the 3900–2700 cm⁻¹ spectral region is shown for two DMSO/sucrose solutions (S4: 10% v/v DMSO with 0.5 M sucrose, S2: 10% v/v DMSO with 1 M sucrose). Contributions of different water fractions were fitted, as Gaussian profiles centered initially at 3139 cm⁻¹ (fully hydrogen bonded water), 3241 cm⁻¹ (symmetrically hydrogen bonded water), 3389 cm⁻¹ (asymmetrically hydrogen bonded water) and 3533 cm⁻¹ (weakly hydrogen bonded water). The relative shifts in peak positions (C,D) and relative band areas (E,F) of these contributions were determined as a function of the DMSO concentration, in combination with either 0.5 M sucrose (C,E) or 1 M sucrose (D,F).</p

Parameters describing molecular mobility in glasses were determined by fitting DSC data on enthalpy relaxation versus storage.

<p>This was done for solution S1 (5% v/v DMSO, 1 M sucrose, white circles), S2 (10% v/v DMSO, 1 M sucrose, black circles), S3 (5% v/v DMSO with 0.5 M sucrose, white triangles) and S4 (10% v/v DMSO with 0.5 M sucrose, black triangles). In panel A, for S1–4, the natural logarithm of the relaxation time is plotted versus the difference between the storage temperature and T_g. In the panel B, data are presented in Arrhenius plots for deriving activation energies. The dotted line indicates −80°C.</p

Storage stability of PC liposomes with trapped CF, frozen in DMSO/sucrose solutions and stored at different temperatures.

<p>Cryoprotective solutions tested were: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles) HEPES buffered solution without further supplements (grey squares) served as a control. Samples were stored at −150°C (A), −80°C (B) and −25°C (C) for up to 3 months. As a measure for storage stability, CF-retention (i.e. protection against membrane leakiness) was assessed and plotted versus the storage duration. The insets in the panels (A) and (B) show the CF-retention after 90 d at −150°C and −80°C, respectively (no significant differences in CF-retention were found among the formulations). Data points representing mean values ± standard deviations were calculated from four measurements (control samples were measured once).</p

CF leakage rates of PC liposomes with trapped CF in DMSO/sucrose solutions at different subzero temperatures.

<p>The difference between the storage temperature and T_g was plotted against the CF leakage rate. Cryoprotective solutions that were tested: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles).</p

Enthalpy relaxation behavior of DMSO/sucrose solutions (S1–4) at different temperatures below the T_g of the solutions.

<p>DSC pans with solution were maintained ~0–15°C below T_g for up to 300 min, after which thermograms were recorded (A–D). Enthalpy relaxation is evident as an endothermic event (oriented upwards) on top of the glass transition event, increasing with storage duration while decreasing if further from T_g. The area of this event, ΔH_relaxation, was determined and plotted versus the storage duration (E–G), for the indicated storage temperatures. Solution S1 was composed of 5% v/v DMSO, 1 M sucrose (A,E), S2 of 10% v/v DMSO, 1 M sucrose (B,F), S3 of 5% DMSO, 0.5 M sucrose (C,G), and S4 of 10% v/v DMSO with 0.5 M sucrose D,H).</p

Transcript variants detected in the equine <i>PLCz1</i>.

<p>Variants were detected in cDNA sequences of testis tissues from six Hanoverian stallions. The size of the untranslated exon 1, mRNA and coding sequence (CDS) in base pairs, the location of the premature termination codon (PTC) and the predicted number of amino acids are given.</p><p>Transcript variants detected in the equine <i>PLCz1</i>.</p

Genome-Wide Association Study Identifies <i>Phospholipase C zeta 1 (PLCz1)</i> as a Stallion Fertility Locus in Hanoverian Warmblood Horses

<div><p>A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within <i>phospholipase C zeta 1 (PLCz1)</i>. In the close neighbourhood to <i>PLCz1</i> is located <i>CAPZA3</i> (<i>capping protein (actin filament) muscle Z-line</i>, <i>alpha 3</i>). The gene <i>PLCz1</i> encodes a protein essential for spermatogenesis and oocyte activation through sperm induced Ca²⁺-oscillation during fertilization. We derived equine gene models for <i>PLCz1</i> and <i>CAPZA3</i> based on cDNA and genomic DNA sequences. The equine <i>PLCz1</i> had four different transcripts of which two contained a premature termination codon. Sequencing all exons and their flanking sequences using genomic DNA samples from 19 Hanoverian stallions revealed 47 polymorphisms within <i>PLCz1</i> and one SNP within <i>CAPZA3</i>. Validation of these 48 polymorphisms in 237 Hanoverian stallions identified three intronic SNPs within <i>PLCz1</i> as significantly associated with EBV-PAT. Bioinformatic analysis suggested regulatory effects for these SNPs via transcription factor binding sites or microRNAs. In conclusion, non-coding polymorphisms within <i>PLCz1</i> were identified as conferring stallion fertility and <i>PLCz1</i> as candidate locus for male fertility in Hanoverian warmblood. <i>CAPZA3</i> could be eliminated as candidate gene for fertility in Hanoverian stallions.</p></div

Chromatograms of transcripts detected in equine <i>PLCz1</i>.

<p>(A) The sequences of all animals tested show both adjacent splice sites of the cDNA. Both, forward and reverse sequences show ambiguous double traces when elongation passes exon boundary. (B, C) Visualization of single trace sequence is used to portray each single transcript. (B) For the transcripts without a premature termination codon (PTC), exon 4a starts with the residues GAT, (C) whereas PTC-containing transcript variants harbor an extended exon 4b and start with the residues TAG. (D) Genomic sequence was used to illustrate usage of adjacent acceptor splice sites AG-TA for PTC-containing transcripts and AG-GA for non-PTC-containing transcripts. Vertical lines mark the exon/exon boundary or intron/exon boundary, respectively. The black open boxes indicate specific nucleotide residues used.</p

Comparison of equine <i>PLCz1</i> transcript variants with and human <i>PLCz1</i>.

<p>Transcript variants (1) ENSECAT00000012304, (2) ENSECAT00000012372, (3) XM_001497766.3, (4) JX545317, (5) JX545319, (6) ENST00000318197, (7) NR_073075.1, (8) JX545318, and (9) JX545320 are presented. (1–4) Human and equine variants without a premature termination codon (PTC) in sequences are indicated. (5–9) A PTC is activated within human and equine variants at similar positions (c.136). The PTC region is highlighted by a red box.</p