Impact of high fat diet on the sterol regulatory element-binding protein 2 cholesterol pathway in the testicle.

Male fertility has been shown to be dependent on cholesterol homeostasis. This lipid is essential for testosterone synthesis and spermatogenesis, but its levels must be maintained in an optimal range for proper testicular function. In particular, sperm cells' development is very sensitive to high cholesterol levels, noticeably during acrosomal formation. The aim of this work was to study whether the molecular pathway that regulates intracellular cholesterol, the sterol regulatory element-binding protein (SREBP) pathway, is affected in the testicles of animals under a fat diet. To investigate this, we took advantage of the non-obese hypercholesterolemia (HC) model in New Zealand rabbits that displays poor sperm and seminal quality. The testicular expression of SREBP isoform 2 (SREBP2) and its target molecules 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and low-density lipoprotein receptor (LDLR) were studied under acute (6 months) and chronic (more than 12 months) fat intake by RT-PCR, western blot and immunofluorescence. Our findings showed that fat consumption promoted down-regulation of the SREBP2 pathway in the testicle at 6 months, but upregulation after a chronic period. This was consistent with load of testicular cholesterol, assessed by filipin staining. In conclusion, the intracellular pathway that regulates cholesterol levels in the testicle is sensitive to dietary fats, and behaves differently depending on the duration of consumption: it has a short-term protective effect, but became deregulated in the long term, ultimately leading to a detrimental situation. These results will contribute to the understanding of the basic mechanisms of the effect of fat consumption in humans with idiopathic infertility.

Implication of vitamin A deficiency on vascular injury related to inflammation and oxidative stress. Effects on the ultrastructure of rat aorta.

BACKGROUND: Vitamin A deficiency induces activation of NF-kB and impairs activities of antioxidant enzymes in aorta. AIM OF THE STUDY: We study the effect of vitamin A deficiency on the aorta histoarchitecture and the possibly contribution of its prooxidant and inflammatory effects to artery alterations. METHODS: Twenty-one-day-old Wistar male rats were fed during 3 months with vitamin A-deficient diet (-A, n = 8) or the same diet containing 8 mg of retinol palmitate/kg of diet (+A, control, n = 8). In aortas, thiobarbituric reactive substances and reduced glutathione levels were measured by spectrophotometry. Expressions of TNF-alpha, NOX-2, VCAM-1, and TGF-beta1 were assessed by RT-PCR and Western Blot. The morphology of aorta was examined by light and transmission electron microscopy. RESULTS: In -A rats, high levels of TBARS in serum and aorta and low levels of GSH in aorta were found. An increased expression of TNF-alpha, NOX-2, VCAM-1, and TGF-beta1 in aorta from -A rats was observed. Examination of the intimal layer by light microscopy indicated the presence of an irregular surface in -A aortas. TEM studies showed large vacuoles and multivesicular bodies along the endothelium and also multivesicular bodies in the subendothelial space of aortas from -A rats. Furthermore, the histological appearance of internal elastic lamina was different from control. Small vesicles in the medial layer were observed in aortas from vitamin A-deficient rats. CONCLUSIONS: Vitamin A deficiency produces histoarchitectural alterations in aorta, which can be associated, at least in part, to the oxidative stress and inflammation induced by vitamin A deficiency.

Protein fraction isolated from epididymal fluid re-associates sperm in vitro: possible role of serpins in rat rosettes assembly.

In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an alpha-1 antitrypsin and a new protein with an alpha-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm-proteases interaction was proposed.

Inhibition of early endosome fusion by Rab5-binding defective Ras interference 1 mutants.

Rin1 has been shown to play an important role in endocytosis. In this study we demonstrated that depletion of Rin1 from the cytosol blocked the fusion reaction. More importantly, endosome fusion was rescued by the addition of Rin1 proteins depending on the presence of Rab5, and its effector EEA1. Furthermore, we found that Syntaxin 13, but not Syntaxin 7, was required by Rin1 to support endosome fusion. We also identified six mutations on the Vps9 domain of Rin1 that failed to rescue the fusion reaction. Two of them, Rin1: D537A and Rin1: Y561F mutants showed dramatic inhibitory effect on the fusion reaction, which correlate with their inability to properly activate Rab5 or to bind endosomal membranes. Taken together, our results suggest that specific residues on the Vsp9 domain of Rin1 are required for its interaction with Rab5, binding to the endosomal membranes and subsequent regulation of the fusion reaction.

Sublethal concentrations of chlorpyrifos induce changes in the thermal sensitivity and tolerance of anuran tadpoles in the toad Rhinella arenarum?

Amphibians are considered one of the groups most susceptible to chemical contamination, therefore are good bio-indicators of aquatic pollution. Synergistic effects of temperature and pesticides have been found in amphibians determining amplified toxicity effect on survival and malformations with increasing temperatures. We studied the sensitivity of sublethal concentrations of chlorpyrifos in Rhinella arenarum tadpoles over on two fitness related thermal traits: locomotor swimming performance and thermal tolerance limits (CTmax = critical thermal maximum and CTmin = critical thermal minimum). Our result shows a decrease in the locomotor performance of R. arenarum tadpoles with increasing sublethal chlorpyrifos concentrations. The experimental temperature increased locomotor performance but this being only significant for the control whereas tadpoles raised at any sublethal chlorpyrifos concentration did not increase their total swimming distance with temperature (Concentration × Temperature interaction, P < 0.019). Chlorpyrifos toxicity decreases maximum swimming distance but this reduction not compensated at high temperatures that do enhance swimming performance in the control treatment. On the other hand, higher chlorpyrifos sensitivity in CTmax than CTmin since tadpoles exposed to all polluted treatments exhibits a significant decline in CTmax but not in CTmin. Current global warming and the increase of atypical climatic events, such as heat waves may put at risk the larval chlorpyrifos polluted populations of R. arenarum. Our results show that the sublethal concentrations of the chlorpyrifos pesticide may affect the fitness and survival of the larvae of R. arenarum.

Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis.

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.

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.

Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa.

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility. ABBREVIATIONS: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation ANOVA: analysis of variance.

A Mouse Model of Diet-Induced Obesity Resembling Most Features of Human Metabolic Syndrome.

Increased chicken-derived fat and fructose consumption in the human diet is paralleled by an increasing prevalence of obesity and metabolic syndrome (MS). Herein, we aimed at developing and characterizing a mouse model of diet-induced obesity (DIO) resembling most of the key features of the human MS. To accomplish this, we fed male C57BL/6J mice for 4, 8, 12, and 16 weeks with either a low-fat diet (LFD) or a high-chicken-fat diet (HFD) and tap water with or without 10% fructose (F). This experimental design resulted in the following four experimental groups: LFD, LFD + F, HFD, and HFD + F. Over the feeding period, and on a weekly basis, the HFD + F group had more caloric intake and gained more weight than the other experimental groups. Compared to the other groups, and at the end of the feeding period, the HFD + F group had a higher adipogenic index, total cholesterol, low-density lipoprotein cholesterol, fasting basal glycemia, insulin resistance, hypertension, and atherogenic index and showed steatohepatitis and systemic oxidative stress/inflammation. A mouse model of DIO that will allow us to study the effect of MS in different organs and systems has been developed and characterized.

Semen quality and sperm function loss by hypercholesterolemic diet was recovered by addition of olive oil to diet in rabbit.

Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality.

Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.

Regulated serine proteinase lytic system on mammalian sperm surface: there must be a role.

Serine proteases play key roles in many biological processes, regulating surface proteins that are key-points in signaling pathways. Several studies have reported the presence of members of this protease family in sperm from various species. The precise regulation of their activity is thought to be performed by specific endogenous or extrinsic inhibitors. The contribution of the sperm serine to proteases to fertilization has been demonstrated by synthetic inhibitors and several single knock out experiments, but to date, there is no evidence that links a single enzyme to a single step of fertilization. The explanation for the failure in the understanding of the "one-enzyme-one-process" hypothesis may be that sperm have multiple serine proteases as a mechanism to ensure the success of fertilization. In addition to the classical purification and expression studies, we summarized recent advances in proteomics and performed a bioinformatics search of proteases and inhibitors, providing support for the idea of redundancy. This review summarizes current knowledge about serine proteases and their inhibitors in sperm capacitation and maturation, identifies questions that need to be answered, and provides a reference for future research.

Effect of climatic variables on seasonal morphological changes in the testis and epididymis in the wild rodent Microcavia australis from the andes mountains, Argentina.

It has been shown that seasonal changes, especially in arid areas have a large influence on gonadal changes of the species that inhabit these areas. We studied a wild hystricomorph Microcavia australis in its natural habitat in the arid Andes Mountains. Sampling of adult males was carried out every 2 months. After autopsy, testes and epididymides were weighed and processed to obtain histological samples. Testes were analyzed with a microscope to measure seminiferous tubule area and diameter for each sampled month. Epididymides were used to determine spermatozoon storage in the cauda region. Results illustrate morphological changes in the testis and epididymis along the year. A high output of sperm cells was detected from middle winter to middle summer and a complete shutdown of spermatogenesis at the end of summer. The initiation of testicular activity coincided with month with the shortest day length, in dry season and very low temperature. On the other hand, gonadal regression started in the middle of summer with long day length, in the wet season, and high temperatures. Rainfall, temperature, and day length seem to be important for the testis cycle. We suggest that photoperiod could be a good predictor for an oncoming period suitable for breeding, and males may probably use it as a signal to regulate gonadal activity.

Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction.

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm-oocyte interactions.

Ceramides with 2-hydroxylated, very long-chain polyenoic fatty acids in rodents: From testis to fertilization-competent spermatozoa.

Sphingolipids from rodent testis and spermatozoa are known to contain non-hydroxylated (N-) and 2-hydroxylated (2-OH) very-long-chain polyunsaturated fatty acids (VLCPUFA). In this study, the contribution of species with each type of fatty acids to the total ceramides (Cer) and sphingomyelins (SM) was investigated in rat and mouse testis and in rat spermatozoa. The major VLCPUFA in both lipids of testis were N- and 2-OH versions of 28:4n-6, 30:5n-6 and 32:5n-6 in the rat, and predominantly of 30:5n-6 in the mouse. Absent altogether from rat pre-puberal testes, SM and Cer with N-VLCPUFA appeared 10 days earlier than those with 2-OH VLCPUFA in postnatal development, in association with germ cell differentiation. Conversely, in adult fertile rats that were gradually deprived of germ cells in vivo after treatment with doxorubicin, SM and Cer with N-VLCPUFA decreased earlier than their 2-OH counterparts, and neither was present in aspermatogenic testes. In rat epididymal spermatozoa, the content of Cer prevailed over that of SM and 2-OH VLCPUFA prevailed over N-VLCPUFA in both lipids. In mature gametes, the acrosomal reaction resulted in an almost complete hydrolysis of the species of SM that contain both types of VLCPUFA to produce the corresponding Cer. Ceramides are biosynthetic precursors of SM in the testis, but themselves final products in spermatozoa. VLCPUFA-rich SM and Cer are thus produced in germ cells with the teleological objective of fulfilling their ultimate physiological role in spermatozoa that are apt and ready to fertilize an oocyte.

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.

Segregation of micron-scale membrane sub-domains in live murine sperm.

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.

Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: its role in in vitro fertilization.

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.

Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5 of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2 SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25 of total proteins respectively. The bicarbonate buffer solubilized 45, and the detergents 55 and 65 respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50 of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.