Hypercholesterolemia Impaired Sperm Functionality in Rabbits

et al. (2010) Hypercholesterolemia Impaired Sperm Functionality in Rabbits. PLoS ONE 5(10): e13457. doi:10.1371/journal.pone.0013457 Hypercholesterolemia Impaired Sperm Functionality in Rabbits Tania E. Saez Lancellotti 0 Paola V. Boarelli 0 Maria A. Monclus 0 Maria E. Cabrillana 0 Marisa A. 0 Clementi 0 Leandro S. Espnola 0 Jose L. Cid Barra 0 Amanda E. Vincenti 0 Analia G. Santi 0 Miguel W. 0 Forne s 0 Colin Combs, University of North Dakota, United States of America 0 1 Laboratorio de Investigaciones Androlo gicas de Mendoza (LIAM), Instituto de Histolog a y Embriolog a de Mendoza (IHEM), Facultad de Ciencias Me dicas, Universidad Nacional de Cuyo - Centro Cient fico Tecnol o gico (CCT), Mendoza - Consejo Nacional de Investigaciones Cient ficas y Te cnicas (CONICET) , Mendoza , Argentina , 2 Facultad de Ciencias Me dicas, Instituto de Investigaciones, Universidad del Aconcagua, Mendoza, Argentina, 3 Laboratorio de Servicios y Ensayos, Instituto Nacional de Tecnolog a Industrial (INTI)-Frutas y Hortalizas , Luja n de Cuyo, Mendoza , Argentina 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. - Funding: This work was supported by PICT (Proyecto de Investigacion Cientifica y Tecnologica) 15 32925, Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCyT); PICTO (Proyecto de Investigacion Cientifica y Tecnologica Orientado) 00116-2007 ANPCyT; Secyt 06/J307(Secretaria de Ciencia y Tecnologica)-National University of Cuyo and CIUDA (Consejo de Investigaciones de la Universidad del Aconcagua)-Aconcagua University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Cholesterol (chol) is a steroid lipid found in the cell membranes and transported in the blood plasma of all animals. However excessive levels of chol in blood circulation (hypercholesterolemia), are strongly associated with progression of atherosclerosis [1]. Chol is an essential component of mammalian plasma membranes (PM) where it is required to establish proper membrane permeability and fluidity [2]. Within the PM, chol has also been implicated in cell signaling processes [3]. Changes in the organization of membrane lipids can have profound consequences on cellular functions such as signal transduction and membrane trafficking [4,5]. The lipid bilayer of the rabbit sperm membrane, as other mammalian sperm cells, consists mainly of phospholipids and chol, at a molar ratio of 1.5 [6]. Cholesterol is also abundant in other subfractions of rabbit semen (seminal plasma and droplets). Sperm membrane undergoes several modifications from the testis, were they are produced, to the female tract. Membrane lipids, especially chol, are responsible for changes in membrane fluidity and cell responsiveness to the environment, alterations involved in a series of physiological events that are unique for these cells [7]. Chol efflux from PM leads to changes in membrane structure and fluidity that give rise to the sperm capacitated state [8]. Capacitation is defined as the time-dependent acquisition of fertilization competence [9], ability acquired by the sperm during its transit through the female tract. This process involves a PKAregulated increase in tyrosine phosphorylation (p-Y) of a subset of proteins [4,10,11], and is generally assessed as the ability of the acrosome-intact sperm to undergo AR in response to physiological inducers such as the zona pellucida or progesterone [10,12]. Animals fed with saturated fat-enriched diets raise their plasmatic chol levels and this would have impact on the cell-specific lipid equilibrium between chol and phospholipids that organize the PM [13]. The later modification could affect cellular functions as signal transduction pathways coupled to membrane chol. Sperm membrane lipids are highly responsive to dietary manipulation [13]. Chol-rich diets have been shown to produce a decrease in sperm AR kinetics [14], and detrimental effects on Leydig and Sertoli cell secretory capacity in rabbits [15]. Moreover, previous works showed that human male infertility might be associated with altered lipid metabolism in seminal plasma [16]. The present study aimed at investigating the effects of dietinduced hypercholesterolemia on rabbit semen and sperm physiology, membrane cholesterol concentration, cell motility, capacitation and acrosome reaction. Materials and Methods Ethics statement The animal studies described here were reviewed and approved by the animal care and use committees of School of Medicine National University of Cuyo (Institutional Committee for Use of Laboratory Animals, IACUC). Reagents Unless otherwise stated, all chemicals and solvents of the highest grade available were obtained from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Animals and diets For the purposes of this study, twelve fertile male White New Zealand rabbits (1.5 months old of age, acquired fromDon Cipriano Farm, Mendoza, Argentina) were caged individually for 11 months with a photoperiod of 12 hours light/day and a temperature ranging from 1825uC. Animals were fed ad libitum a standard rabbit diet composed of 17% crude protein, 16% fiber, 2% minimal ether e (...truncated)