La dieta alta en grasas y el Aceite de oliva virgen extra (AOVE) modulan la homeostasis lipídica testicular a través de la proteína Srebp-2

La obesidad y el sobrepeso están aumentando en todo el mundo y tienen influencias perjudiciales en varias funciones del cuerpo humano, incluida la salud reproductiva. Definir los mecanismos de cómo el aumento del colesterol en la dieta amenaza la salud reproductiva masculina es fundamental para desarrollar enfoques que mejoren los problemas actuales de fertilidad.Fil: Funes A, . Universidad del AconcaguaFil: Crescitelli, J.. Universidad del AconcaguaFil: Fernandez, M.. Universidad del AconcaguaFil: Monclús, M.. Universidad del AconcaguaFil: Fornés, M.. Universidad del AconcaguaFil: Saez. Lancellotti E. . Universidad del Aconcagu

Asociación de espermatozoides en el epidídimo de rata. Expresión de Serpina 1F, una proteína involucrada en este proceso madurativo.

La asociación epididimaria de espermatozoides es un fenómenoobservado en numerosas especies de mamíferos. Cuando los espermatozoides madurosalcanzan la cola del epidídimo pueden asociarse entre sí mediante sus cabezasformando estructuras características, cuya morfología depende de cada especie. Entrelas cabezas asociadas se describió la presencia de un material electron denso denaturaleza proteica. En la rata estas asociaciones se denominan ”rosetas“. De estemodo, los espermatozoides permanecen almacenados hasta la eyaculación. Una vezdepositados en el tracto reproductor femenino o bien liberados a un medio de cultivo seactiva la movilidad espermática. Esto produce que uno a uno los espermatozoides sedesprendan originando una suspensión de células móviles y aisladas. En las especiesde mamíferos estudiadas, este proceso de asociación reversible se relaciona con elgrado de maduración alcanzado por los espermatozoides. En cuanto a la funciónbiológica se ha propuesto tanto la protección de las gametas frente a una reacciónacrosomal anticipada como la adquisición de una una mayor velocidad dedesplazamiento de las asociaciones lo que supondría ventaja en caso de competenciaespermática. En trabajos previos nuestro grupo aisló e identifico en el fluido epididimariode la región caudal de rata la proteína Serpina 1 F que pertenece a la familia de lasSerpinas (inhibidores de serin proteasas). Luego demostramos que Serpina 1Fparticipaba en la asociación epididimaria de espermatozoides en esta especie medianteensayos de re asociación in vitro

Efecto del aceite de oliva sobre el metabolismo del colesterol en conejos hipercolesterolémicos.

El aceite de oliva, componente esencial de la Dieta Mediterránea, posee efectosbeneficiosos sobre la hipercolesterolemia, principal factor de riesgo de enfermedadescardiovasculares. La administración de una dieta grasa en conejos provoca laacumulación de colesterol en la membrana celular del espermatozoide y se correlacionacon defectos en la biogénesis del acrosoma y de la cabeza en general. Este incrementode colesterol interfiere con el normal funcionamiento de la gameta masculina y podría sercausa de infertilidad masculina. Interesantemente, la administración de aceite de oliva aestos animales mejoró los parámetros alterados por la ingesta grasa.Nuestro interés reside en estudiar la vía intracelular de colesterol en testículo deconejos bajo diferentes dietas experimentales. El efecto beneficioso del aceite de oliva anivel celular no ha sido estudiado a nivel celular/molecular

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 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

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