Manchette-acrosome disorders and testicular efficiency decline observed in hypercholesterolemic rabbits are recovered with olive oil enriched diet.

High-fat diet is associated with hypercholesterolemia and seminal alterations in White New Zealand rabbits. We have previously reported disorders in the development of the manchette-acrosome complex during spermiogenesis and decreased testicular efficiency in hypercholesterolemic rabbits. On the other hand, olive oil incorporated into the diet improves cholesterolemia and semen parameters affected in hypercholesterolemic rabbits. In this paper, we report the recovery-with the addition of olive oil to diet-from the sub-cellular mechanisms involved in the shaping of the sperm cell and testicular efficiency altered in hypercholesterolemic rabbits. Using morphological (structural, ultra-structural and immuno-fluorescence techniques) and cell biology techniques, a reorganization of the manchette and related structures was observed when olive oil was added to the high-fat diet. Specifically, actin filaments, microtubules and lipid rafts-abnormally distributed in hypercholesterolemic rabbits-were recovered with dietary olive oil supplementation. The causes of the decline in sperm count were studied in the previous report and here in more detail. These were attributed to the decrease in the efficiency index and also to the increase in the apoptotic percentage in testis from animals under the high-fat diet. Surprisingly, the addition of olive oil to the diet avoided the sub-cellular, efficiency and apoptosis changes observed in hypercholesterolemic rabbits. This paper reports the positive effects of the olive oil addition to the diet in the recovery of testicular efficiency and normal sperm shaping, mechanisms altered by hypercholesterolemia

Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model.

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic rabbit model is a useful tool to study serum cholesterol increment linked to sub/infertility

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

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

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