Estudio del impacto de las dietas hiperlipémicas en células mononucleares en sangre periférica: resultados preliminares

Los trastornos metabólicos asociados con la dieta tienen un gran impacto en la salud, debido a su relación con enfermedades crónicas no transmisibles. Las células mononucleares de sangre periférica (PBMC) tienen un papel importante como biomarcadores tempranos en el estudio del impacto de las dietas ricas en grasas en el metabolismo de los lípidos. Estos permiten profundizar el conocimiento de la patogénesis por métodos no invasivos. Por lo tanto, el objetivo es estudiar PBMC como una herramienta de investigación para la expresión génica en las alteraciones del metabolismo de los lípidos. Se obtuvieron muestras de diez conejos neozelandeses, divididos en el grupo control (n=5) alimentado con alimento balanceado (C), y el grupo de casos (n=5) alimentado con el mismo alimento suplementado con un 17% de grasa bovina (G). Los grupos de grasa no reciben sobrecarga de fructosa, manteniendo constante la concentración de carbohidratos y proteínas, típica de los alimentos básicos equilibrados. Se realizaron pruebas bioquímicas para determinar los niveles de glucosa en sangre (Gl), triglicéridos (TG), colesterol total (CT) y lipoproteínas de alta densidad (HDL). En PBMC, se realizaron pruebas inmunohistoquímicas para SREBP1c y SERBP2 (unión de proteínas a elementos reguladores estériles). Valores similares de Gl (C: 140 ± 28.4 mg / dL vs G: 118.3 ± 12.0 mg / dL) y TG (C: 144.1 ± 15.5 mg / dL vs.G: 135.6 ± 8.3 mg / dL) pueden observarse en estudios bioquímicos preliminares de ambos grupos, mientras que el grupo G muestra un aumento en CT (42.8 ± 21.6 mg / dL) en comparación con el grupo C (27.1 ± 4.5 mg / dl). Los valores de HDL fueron menor en el grupo C (9,3 ±4,76 vs 11,8 ± 3,45 mg/dl), y se calculó el índice aterogénico, con un valor mayor en grupo G (3,64 vs 2,91). Sin embargo, algunos animales del grupo G tienen valores similares al grupo C para CT (21.7 ± 2.4 mg / dl), HDL (9,9 ± 3,8) e IA (2,2), grupo de normocolesterolemia (NC). Por lo tanto, estos animales no muestran cambios bioquímicos a pesar de la ingesta de grasas como ocurre con otros. Los estudios de tejido hepático mostraron esteatosis (tinción con aceite rojo O), así como la presencia de SREBP1c (relación perinuclear / nuclear: C: 2.0, F: 0.71, NG: 1.81) y SERBP2 (relación perinuclear / nuclear: C: 3.29, F: 0.85, NG: 0.57) en PBMC. En conclusión, estos resultados indicarían una activación de la regulación génica sin cambios a nivel bioquímico en grupo NC. Estos resultados indican que es posible estudiar la expresión génica en PBMC, porque se puede observar la presencia de moléculas específicas relacionadas con el metabolismo de los lípidos.Fil: Avena, M. V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad "Juan Agustín Maza"; ArgentinaFil: Elias, María. Universidad "Juan Agustín Maza"; ArgentinaFil: Heredia, Rocío. Universidad "Juan Agustín Maza"; ArgentinaFil: Mussi Stoizik, Jessica Anabella. Universidad "Juan Agustín Maza"; ArgentinaFil: Colombo, Regina Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Funes, Abi Karenina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Fornes, Miguel Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Saez Lancellotti, Tania Emilce Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Boarelli, Paola Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad "Juan Agustín Maza"; ArgentinaIV Reunión Conjunta de Sociedades de Biología de la República Argentina: “Nuevas Evidencias y Cambios de Paradigmas en Ciencias Biológicas”MendozaArgentinaSociedad de Biología de CuyoSociedad de Biología de CórdobaAsociación de Biología de Tucumá

Hypercholesterolemia Impaired Sperm Functionality in Rabbits

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events

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

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

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

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

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

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

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

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