Histological alterations observed by light and electron microscopy.

<p>Light Microscopy (LM): Seminiferous tubule cross-sections of NCR (A and C) and HCARDA (B and D). Normal evolution from spermatogonium to sperm cells (A) and distinctive cells from VIII stage (C) are observed. An acrosomic granule is well formed within the Golgi vesicle in NCR(C, arrow). In HCARDA; it was detected: empty holes (lipid droplets, B, asterisks); abnormal development of sperm head (D, arrow head); round spermatids with a big vacuole close to the nucleus and abnormal Golgi features (D, arrow)and elongated spermatids with asymmetric and flexuous nucleus (D, arrowhead). 400X (A and B) and 620X (C and D). Electron Microscopy (EM): Ultrastructure of acrosome development and nucleus shaping of NCR (A, C and E) and HCARDA (B, D, F and G). The acrosomal granule was observed centrally located within the Golgi vesicle (A, arrow). Perinuclear ring of the manchette is indicated (C, asterisk). The microtubule mantle of the manchette was observed parallelly assembled (E, bold parallel arrows) and symmetrically distributed from the central axis (see acrosomal asymmetry measurement in materials and methods). In HCARDA, it was observed: misshapen and asymmetrical proacrosomal vesicle with narrow and expanded zones (B, #); membrane whorls inside spermatogenic cells (D, arrowheads); membranous vacuoles beside the acrosome (F, +); curved sperm heads with non-parallel assembled manchette microtubules (G, unparallel arrows). Magnifications: A, B: 10000X; C, D: 40000X; E, F, G: 20000X.</p

Manchette organization during spermiogenesis.

<p>Spermatogenic cells were isolated, stained and ordered according acrosome maturation (Early to Late). Spermatid nuclei were stained red with propidium iodide (A, F, K, P, U, Z) and manchette microtubules were visualized by anti-Alpha tubulin (green, B, G, L, Q, V, AA). In NCR, it was visualized: a polarized manchette (G, Mz: Manchette zone, H) in opposite location to the acrosome (G, Az: Acrosome zone, H) and condensed nuclei (K, M). Instead, a diffused manchette (Q, V, AA), abnormal nucleus condensation (P, U, Z, white arrow) and persistency of residual bodies (AA, BB, CC, asterisks) were visualized in HCARDA. Dashed line indicates the central axis. Phase-contrast microscopy images of the corresponding immunofluorescence images are included (DIC). Last column shows stained cells resembling the same stadium. <i>n</i> = 100 cells. Magnification:650X.</p

Morphological alterations in isolated spermatogenic cells and acrosomal asymmetry (Asymmetry Index).

<p>Isolated cells were arranged from round (left) to elongated spermatids (right) following successive steps of transition from Golgi to acrosome in NCR (A to I, upper row) and HCARDA (J to R, lower row). Dashed line denotes the central axis(C and N); asterisks indicate acrosomal ends (N); arrow points the nuclear ring position (G) and abnormal vacuole is marked with + (N). 650X. <b>Asymmetry Index</b> (S): Distance from the central axis to each acrosomal end in isolated cells (<i>n</i> = 30 cells per condition) was calculated and expressed as asymmetry index (NCR: ■, HCARDA: Δ). A major index corresponds to higher asymmetry (<i>p</i> ≤ 0.003).</p

Morphological changes in seminal sperm.

<p>Representative sperm cells isolated from semen of NCR (left panel) and HCARDA (right panel) rabbits. Four sperm heads from NCR show the slight variance among the normal sperm population found (A, B, C, D). Instead, several abnormalities were observed in HCARDA: acrosomal lost (E), cytoplasmic droplet persistence (F), tapered head (G) and asymmetry in the implantation of the tail (H). 1000X. Cells with head and tail defects from NCR (■) and HCARDA (Δ) were quantified and are represented as the mean ± SD of the ratio between the number of alterations / 100 sperm cells counted in thirty different cells. The experimental time is represented in x axis since the beginning of ED. Percentages were significantly different (<i>p</i> ≤ 0.05) from six months of ED.</p

Manchette (microtubules), GM1 (raft membrane micro domains) and actin filaments arrangement during spermiogenesis.

<p>Spermatogenic cells were isolated, stained and observed under fluorescence microscopy. Manchette microtubules were detected with anti alpha-tubulin (green, B, F, J, N), actin filaments were localized with anti alpha-actin (red, I, M, Q, U) and GM1 sphingolipids were detected by cholera toxin beta subunit (red, A, E; green, R, V). In control cells, the manchette was polarized (NCR; B, J) and co-localized with GM1 (NCR; C—Mz), opposite to the acrosome (K, Az). Instead, alpha-tubulin and GM1 were diffused and without co-localization in cells isolated from HCARDA (E, F, G). Actin filaments were localized with alpha-tubulin in the manchette (I, J, K—Mz) in NCR. But in HCARDA, actin and tubulin were visualized diffused (M, N, O). Interestingly, actin and GM1 were localized in the manchette in NCR (Q, R, S—Mz) but were seen dispersed in HCARDA (U, V, W). Phase-contrast microscopy images of the corresponding immunofluorescence images are included (DIC). Last column shows stained cells resembling the same stadium. Mz: manchette zone; Az: acrosomal zone. <i>n</i> = 100 cells. Magnification: 650X.</p

General characteristics of fresh rabbit semen samples (mean ± SEM).

*<p>p<0.05, n = 25.</p

Experimental groups.

<p>Twenty New Zealand rabbits were initially distributed at random in three groups: NCR, HCR, OO at 5 months of age (corresponding to experimental time (et) = 0 months). After 4 months, NCR continued with normal diet (ND) and HCR was split in two subgroups: HCR and ½ HCR. HCR continued with experimental diet 1 (ED1) and the last group was fed with ED2. Four months later (et = 8 m) ½ HCR was divided again in two (subgroup II): ½ HCR (fed with ED2) and ½ HCR+½ OO (fed with ED3). m = months, n = number of experimental animals. More details in the text.</p

Impact of diet on sperm functionality: membrane response to hypo-osmotic swelling test.

<p>Bars represent the percentage (mean ± SEM) of spermatozoa swollen from NCR (black bar), OO, HCR, ½ HCR and ½ HCR+½ OO (white bars). The experiment was performed at least three times with each animal. n = 25 samples. *** = significantly different from NCR (p<0.001).</p

Impact of diet on sperm functionality: sperm capacitation and acrosome reaction.

<p>Protein tyrosine phosphorylation (A) and acrosomal exocytosis index (B) of rabbit spermatozoa. A: phospho-Y proteins from control (NCR), HCR and ½ HCR+½ OO showed different patterns ranging from one band (non-capacitated, culture medium without BSA (−), approximately 60 kDa) to many bands (capacitated with BSA (+), from over 20 to 100 kDa). Notice that capacitated sperms from HCR decreased the p-Y protein pattern compared with NCR, and ½ HCR+½ OO shows a p-Y pattern resembling control (NCR) conditions. The experiment was performed at least three times and representative blot is shown. B: Bars represent AR index of spermatozoa from NCR, ½ HCR, ½ HCR+½ OO and OO after 10 µM progesterone incubation. AR index corresponds to normalized data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052386#s2" target="_blank">Materials and Methods</a>). n = 25 samples. *** = significantly different from NCR (p<0.001).</p

Effect of dietary treatment on sperm membrane cholesterol.

<p>Fluorescence micrographs showing cholesterol content in plasma membrane of ejaculated rabbit spermatozoa detected by filipin probe. Images correspond to filipin-stained sperm cells (×630) from NCR (A), OO (B) HCR (C), ½ HCR (D) and ½ HCR+½ OO (E). Compare the strong signal detected in HCR (C) with the one from the ½ HCR+½ OO (E). The experiment was performed at least three times with 20 sperm from each animal.</p