Precocious capacitation and increased spontaneous acrosome reaction in sperm from <i>DefbΔ9/DefbΔ9</i> mice.

<p>Figure 4A: FITC-conjugated Pisum sativum (PSA) lectin labelling of acrosome reacted sperm. Percentage of spontaneous AR in wild-type (+/+) and <i>DefbΔ9/DefbΔ9</i> (−/−) sperm determined by PSA-FITC lectin labelling before (T0) and after 90 minutes incubation (T90) in complete capacitation medium (mean ±SD; n = 4). *, p<0.05. Figure 4B: Zonadhesin antibody binding and quantification. Images show representative ZAN antibody binding to a sperm from <i>DefbΔ9</i> (−/−) male after capacitation, visualised with a goat anti-rabbit IgG conjugated to Alexa Fluor 594 around the sperm head. Left panel (bright field) and right panel (fluorescent image). Lower panel shows percentages of ZAN exposure evaluated on live spermatozoa from +/+ and −/− before (T0) and after (T90) incubation in capacitation medium. Figure 4C: Acrosome integrity following Coomassie blue G250 staining of fixed sperm. Right panel shows representative image of sperm that is acrosome reacted (arrow) lacking staining and other non-AR sperm with intense staining. Left panel graph shows average percentage of acrosome reacted sperm from three independent experiments where over 150 sperm were counted per sample before and after incubation in complete capacitation medium (mean ± SD; n = 3). p<0.03. Figure 4D: Percentage of capacitation evaluated by the ability of sperm to undergo the AR. AR induced by 10 µM calcium ionophore A23187 in spermatozoa from mutant (−/−) and wild-type (+/+) animals and level of PSA-FITC used to determine AR directly after the treatment (T0) and 90 minutes after (T90) (mean ±SD; n = 4). *, p<0.01. Figure 4E: Sperm-egg binding assay. Light microscopy images show cumulus-free eggs from superovulated CD1 females with sperm from wild-type animals (+/+) (upper left panel) and no sperm from <i>DefbΔ9−/−</i> males bound to the eggs (upper right panel). Sperm were also incubated with 2-cell embryos as a control for non-specific binding (left egg in upper left panel). A range of 47–86 eggs per genotype were used for each set of experiments (n = 3). Original magnification ×20. Graph shows comparison of the average number of sperm from wild-type (+/+) and <i>DefbΔ9</i> (−/−) males bound eggs following 45 minute incubation (mean ± SD; n = 3). **, p<0.001.</p

<i>DefbΔ9/DefbΔ9</i> male mice have more fragile sperm with reduced motility.

<p>Figure 3A: Cauda epididymal sperm from <i>DefbΔ9/DefbΔ9</i> (−/−) show an increased number of detached heads compared to wild-types (+/+). Photomicrographs represented are in phase-contrast microscopy at original magnification of ×40. Fragility of the mutant vs wild-type derived sperm isolated from cauda was determined by dropping the suspensions onto a glass slide and analysing the number of detached heads (arrowheads). A total of 200 sperm were analysed for each slide, which represented one animal. An average of 3 pairs was analysed (mean ± SD; n = 3). p = 0.048. Figure 3B: Spermatozoa from <i>DefbΔ9/DefbΔ9</i> mice have reduced motility. Percentage of total (left) and progressive (right) spermatozoa motility in <i>DefbΔ9</i> +/+ and <i>DefbΔ9</i> −/− mice using CASA before (time 0 mins, T0) and after (time 90 mins, T90) sperm capacitation (mean ±SD; n = 4). *, p<0.001.</p

Ultrastructure of spermatozoa from cauda, caput and testis from wild-type littermates (+/+) and <i>DefbΔ9/DefbΔ9</i> (−/−) male mice reveals a defect in microtubule structure.

<p>Figure 5A: Transmission Electron Microscopy (TEM) of sperm from cauda, caput and testis from <i>DefbΔ9/DefbΔ9</i> (−/−) and wild-type littermates (+/+) mice. Top and middle horizontal panels show overviews of cross section of sperm from cauda of +/+ and −/− mice respectively at various levels of the tail (a). Higher magnification of cross section of principal piece (b–d) and mid-piece (e–f) of sperm tails. Upper panel (f) shows normal axoneme (9+2 microtubules, MT), mitochondrial sheath (M), outer dense fibres (ODF) in wild-type mouse sperm. Middle panel (f) shows clear disruption and disintegration of the MT (arrowhead) in sperm from <i>DefbΔ9</i> (−/−). Middle panel (b) shows example of additional microtubules (*) other than the classical 9+2 arrangement. Bottom panel shows TEM of caput epididymal (a, b) and testis (c, d) sperm from wild type (+/+) and <i>DefbΔ9</i>(−/−) mice. No obvious microtubule disruptions were observed in sperm within the caput or testis of the mutant mice. Bars: 500 nm and 100 nm as labelled (a–f). Figure 5B: Fluorescent intensity of total intracellular calcium of wild-type (+/+) littermates and <i>DefbΔ9/DefbΔ9</i> (−/−) sperm using Fluo3 AM ester assay. The calcium levels were measured using Fluo-3 AM ester calcium fluorescent indicator at 5 µM concentration incubated with spermatozoa at 20 million/ml for 30 mins at 37°C, samples were washed and loaded onto 96-well plate in duplicates at 100 µl/well, and the plates were read by BMG Labtech FluoSTAR Omega fluorescent reader (mean ± SD; n = 3).*, p<0.002. Values given are after subtracting the background levels of the DMSO controls.</p

Peptide sequence of β-defensins deleted in DefbΔ9 deletion.

<p>Single letter amino acid sequence of the predicted peptide encoded by the β-defensin genes in the <i>DefbΔ9</i> deletion. The signal sequence is separated from the mature peptide sequence as determined by ExPASy proteomics tool <a href="http://web.expasy.org/peptide_cutter/" target="_blank">http://web.expasy.org/peptide_cutter/</a>. These are two exon encoded genes, and the first exon encoded amino acids are in bold, second exon encoded amino acids are non-bold. Mature peptide sequences are aligned using the classical 6 cysteine motif (present in all but Defb50) and spaces are introduced (marked by -) to enable this. Cysteines in the mature peptide are emboldened.</p

<i>DefbΔ9/DefbΔ9</i> male mice are infertile.

<p>Figure 2A: Litter sizes of <i>DefbΔ9/DefbΔ9</i> (−/−) male mice mated to wild-type CD1 females over 3 months. The homozygous mutant males produced no litters. Figure 2B: Litter sizes of DefbΔ9 female mice mated to wild-type CD1 males over 3 months. <i>DefbΔ9</i> (−/−) female mice reproduced normally with no significant difference in litter sizes. The variation in litter size for the wild-type and heterozygous mice between panel A and B is due to the genetic background difference between the females used (CD1 in panel A and C57Bl/6 in panel B). Figure 2C: Histology of testis (left) and epididymis (right) of wild-type (+/+) (top panel) and <i>DefbΔ9/DefbΔ9</i> (−/−) (bottom panel) mice. Tissue from approximately 5 months old mice were fixed in bouin's fixative, paraffin wax blocked, cut at 7 µm thick sections and stained with H&E stain. No obvious histological difference is present between the wild-type and mutant tissue at the light microscopy level. Sperm are easily visible (arrowed) in both wild-type and mutant cauda. Original magnifications ×10.</p