Establishing an association between a peri-operative perfusion score system (PerfSCORE) and post-operative patient morbidity/mortality during CPB cardiac surgery.

BACKGROUND: To date, there is no quality assurance program that correlates patient outcome to perfusion service provided during cardiopulmonary bypass (CPB). A score was devised, incorporating objective parameters that would reflect the likelihood to influence patient outcome. The purpose was to create a new method for evaluating the quality of care the perfusionist provides during CPB procedures and to deduce whether it predicts patient morbidity and mortality. METHODS: We analysed 295 consecutive elective patients. We chose 10 parameters: fluid balance, blood transfused, Hct, ACT, PaO2, PaCO2, pH, BE, potassium and CPB time. Distribution analysis was performed using the Shapiro-Wilcoxon test. This made up the PerfSCORE and we tried to find a correlation to mortality rate, patient stay in the ICU and length of mechanical ventilation. Univariate analysis (UA) using linear regression was established for each parameter. Statistical significance was established when p < 0.05. Multivariate analysis (MA) was performed with the same parameters. RESULTS: The mean age was 63.8 +/- 12.6 years with 70% males. There were 180 CABG, 88 valves, and 27 combined CABG/valve procedures. The PerfSCORE of 6.6 +/- 2.4 (0-20), mortality of 2.7% (8/295), CPB time 100 +/- 41 min (19-313), ICU stay 52 +/- 62 hrs (7-564) and mechanical ventilation of 10.5 +/- 14.8 hrs (0-564) was calculated. CPB time, fluid balance, PaO2, PerfSCORE and blood transfused were significantly correlated to mortality (UA, p < 0.05). Also, CPB time, blood transfused and PaO2 were parameters predicting mortality (MA, p < 0.01). Only pH was significantly correlated for predicting ICU stay (UA). Ultrafiltration (UF) and CPB time were significantly correlated (UA, p < 0.01) while UF (p < 0.05) was the only parameter predicting mechanical ventilation duration (MA). CONCLUSIONS: CPB time, blood transfused and PaO2 are independent risk factors of mortality. Fluid balance, blood transfusion, PaO2, PerfSCORE and CPB time are independent parameters for predicting morbidity. PerfSCORE is a quality of perfusion measure that objectively quantifies perfusion performance.

Corticosterone shifts reproductive behaviour towards self-maintenance in the barn owl and is linked to melanin-based coloration in females.

Trade-offs between the benefits of current reproduction and the costs to future reproduction and survival are widely recognized. However, such trade-offs might only be detected when resources become limited to the point where investment in one activity jeopardizes investment in others. The resolution of the trade-off between reproduction and self-maintenance is mediated by hormones such as glucocorticoids which direct behaviour and physiology towards self-maintenance under stressful situations. We investigated this trade-off in male and female barn owls in relation to the degree of heritable melanin-based coloration, a trait that reflects the ability to cope with various sources of stress in nestlings. We increased circulating corticosterone in breeding adults by implanting a corticosterone-releasing-pellet, using birds implanted with a placebo-pellet as controls. In males, elevated corticosterone reduced the activity (i.e. reduced home-range size and distance covered within the home-range) independently of coloration, while we could not detect any effect on hunting efficiency. The effect of experimentally elevated corticosterone on female behaviour was correlated with their melanin-based coloration. Corticosterone (cort-) induced an increase in brooding behaviour in small-spotted females, while this hormone had no detectable effect in large-spotted females. Cort-females with small eumelanic spots showed the normal body-mass loss during the early nestling period, while large spotted cort-females did not lose body mass. This indicates that corticosterone induced a shift towards self-maintenance in males independently on their plumage, whereas in females this shift was observed only in large-spotted females.

Evolution of the mating system in colonizing plants.

Colonization is likely to be more successful for species with an ability to self-fertilize and thus to establish new populations as single individuals. As a result, self-compatibility should be common among colonizing species. This idea, labelled 'Baker's law', has been influential in discussions of sexual-system and mating-system evolution. However, its generality has been questioned, because models of the evolution of dispersal and the mating system predict an association between high dispersal rates and outcrossing rather than selfing, and because of many apparent counter examples to the law. The contrasting predictions made by models invoking Baker's law versus those for the evolution of the mating system and dispersal urges a reassessment of how we should view both these traits. Here, I review the literature on the evolution of mating and dispersal in colonizing species, with a focus on conceptual issues. I argue for the importance of distinguishing between the selfing or outcrossing rate and a simple ability to self-fertilize, as well as for the need for a more nuanced consideration of dispersal. Colonizing species will be characterized by different phases in their life pattern: dispersal to new habitat, implying an ecological sieve on dispersal traits; establishment and a phase of growth following colonization, implying a sieve on reproductive traits; and a phase of demographic stasis at high density, during which new trait associations can evolve through local adaptation. This dynamic means that the sorting of mating-system and dispersal traits should change over time, making simple predictions difficult.

Sex- and melanism-specific variations in the oxidative status of adult tawny owls in response to manipulated reproductive effort.

Oxidative stress, determined by the balance between the production of damaging reactive oxygen species (ROS) and antioxidant defences, is hypothesized to play an important role in shaping the cost of reproduction and life history trade-offs. To test this hypothesis, we manipulated reproductive effort in 94 breeding pairs of tawny owls (Strix aluco) to investigate the sex- and melanism-specific effects on markers of oxidative stress in red blood cells (RBCs). This colour polymorphic bird species shows sex-specific division of labour and melanism-specific history strategies. Brood sizes at hatching were experimentally enlarged or reduced to increase or decrease reproductive effort, respectively. We obtained an integrative measure of the oxidative balance by measuring ROS production by RBCs, intracellular antioxidant glutathione levels and membrane resistance to ROS. We found that light melanic males (the sex undertaking offspring food provisioning) produced more ROS than darker conspecifics, but only when rearing an enlarged brood. In both sexes, light melanic individuals had also a larger pool of intracellular antioxidant glutathione than darker owls under relaxed reproductive conditions (i.e. reduced brood), but not when investing substantial effort in current reproduction (enlarged brood). Finally, resistance to oxidative stress was differently affected by the brood size manipulation experiment in males and females independently of their plumage coloration. Altogether, our results support the hypothesis that reproductive effort can alter the oxidative balance in a sex- and colour-specific way. This further emphasizes the close link between melanin-based coloration and life history strategies.

Effects of female postmating odour on male sexual behaviour, in Heliconius butterflies /

The effects of the female postmating odour on male sexual behaviour were examined in Heliconius erato and H. charithonia (Lepidoptera: Nymphalidae). Predictions from the antiaphrodisiac hypothesis were tested using the two reproductive strategies of these species. Within the pupal mating strategy, results from behavioural experiments quantified and statistically tested dispersal rates of pupal-perched males to the presence of stimuli with and without the postmating odour. Results do not support an antiaphrodisiac function to the postmating odour. Similarly, within the adult courtship strategy, behavioural test results indicate that males do not alter their expenditure of energy in terms of either the duration or frequency of courtship behaviours elicited by females with and without the postmating odour. The data from both experiments did not support the antiaphrodisiac hypothesis for the function of the female postmating odour. A novel hypothesis predicting that the postmating female odour acts as an oviposition-deterring pheromone is presented.

Locomotor sensitization and conditioned place preference for amphetamine in late adolescent and adult male and female rats /

reports, the players did not show an anticipatory rise in either Cortisol or testosterone prior to competition. In addition to the effects of status outcome on hormonal levels, it was also found that these hormonal responses were specific to competition. The athletes in the current study did not demonstrate any hormonal responses to the practice sessions. Last, there were significant differences in pre-game testosterone as well as in selfconfidence, cognitive, and somatic anxiety levels depending on the location at which the status contest took place. Pre-game testosterone and self-confidence levels were significantly higher prior to games played in the home venue. In contrast, pre-game somatic and cognitive anxiety levels were significantly higher prior to games played in the away venue. The current findings add to the developing literature on the relationship between hormones and competition. This was the first study to detect a moderating effect of status outcome on testosterone responses in a team sport. Furthermore, this was also the first study in humans to demonstrate that post-contest Cortisol levels were significantly higher after a loss of status. Last, the current study also adds to the sport psychology literature by demonstrating that pre-game psychological variables differ depending on where the status contest is being held: higher self-confidence at home and higher somatic and cognitive anxiety away. Taken together, the results from the current thesis may have important practical relevance to coaches, trainers and sport psychologists who are always trying to find ways to maximize performance. the cycle. The sex-specific age differences in locomotor responses to amphetamine are not due to gonadal immaturity, as females are cycling at this stage of adolescence. However, age differences may reflect the ongoing maturation of the neural substrates that that are involved in locomotor sensitizing, but not rewarding effects of amphetamine.

Individual differences and factors affecting male behaviour in field crickets

Individual differences in male sexual behav~our and the factors influencing calling behaviour were studied in the field crickets Gryllus 2 integer and Q. veletis. In a large (13m) outdoor arena individually numbered adult male ~~ integer started calling at three to five days of age but thereafter the age of individual G. integer males did not affect nightly calling duration. Calling also did not correlate with individual weight. In this study individual male calling was continuously distributed from 0 hrs. per night to 3.5 hrs. per night, on average. A temporal effect on the number of G. integer males calling was observed. The number of males calling through the night was uniform, but a sharp increase in the number calling was observed in the early morning. No difference in calling times was observed between the night and dawn callers. AlsC)' males calling at dawn usually didnotc'all during the preceeding night. Calling and reproductive success in 1979 demonstrated a negative logarithmic relationship while in the 1980(initial) population a negative linear relationship was observed. No relationship was seen in the 1980 high density population. The ratio of non-callers to callers also affected the mating of individuals in the 1979 and1980(initial) densities:-non~callers (males calling .5 hrs. per night, on average, or less) obtained more females when the population contained a high number of callers, this being a negative logarithmic relationship to, No such relationship was observed in the 1980 high density population. Individual displacement varied nightly and was not correlated to amount of calling or reproductive success of individual G. integer males. G. integer males were displa~ed more when in a higher density in the outdoor arena Male G. integer and G. veletis behaviours were also observed in an indoor arena at different densities and, in G. veletis, with respect to female presence. When females were present in the arena, in G. veletis, male calling was reduced. Males of both species called less, on average, when in ~ higher density, than when they were in a lower density. Male displacement of both species increased on average when in a higher density as compared to displacement in a lower density. Aggression was measured by aggressive call-ing and fighting and was studied in regards to density.G. integer demonstrated less aggression in all but one comparison at higher density. No difference was observed in the ratio of aggressive calling to f.ighting comparison in G. integer. G. veletis demonstrated mixed results. No difference in aggression between densities was observed in comparisons. Less.aggression did occur in higher densities when comparisons invol.ved fighting behaviour. Male behaviour represents a competitive strategy against ot~er males, strategy being defined as a genetic (in part) alternative to other strategies. In this sense, the factors of time, density, male-male aggression, and female presence are conditions demonstrated to affect male behaviour in G. integer and G. veletis. Individual male differences and other considerations suggest that alternative male behaviours are represented by at least two conditional strategies. This possibility, and the transient 'or stable nature of genetic polymorphisms in field cricket behaviour are considered.

Mating behaviours of the field cricket (Gryllus integer) at different levels of male density

The reproductive behaviour of the field cricket, Gryllus integer, was systematically observed in indoor arenas to determine the extent of female Choice and male-male competition at different sex ratios representing two male densities (12:6 and 6:6). The costs and benefits to males and females in those two densities were analyzed according to the theory of the evolution o£ leks. Observations were conducted during the dark hours when most calling occurred since hourly rates of courtship song and mating did not fluctuate significantly over a 24 h period. Female mating rates were not significantly different between densities, therefore males at high densities were not advantaged because of increased female tendencies to mate when social stimulation was increased. Mean rates of acoustical signalling (calling and courtin"g) did not differ significantly between densities. Mean rates of fighting by males at the high density were significantly greater than those of males at the low density. Mating benefits associated with callin~courting and fighting were measured. Mating rates did not vary with rates of calling at either density. Calling was not a prerequisite to mating. Courtship song preceded all matings. There was a significant power fit between male mating and courting rates, and male mating and fighting rates at the low, but not at the high, density. Density differences in the benefits associated with increased courting and fighting may relate, in part, to greater economic defensibility and monopoly of females due to reduced male competition at the low density. Dominant males may be preferentially chosen by females or better able to monopolize mating opportunities than subordinate males. Three criteria were used to determine whether dominant males were preferentially chosen by females. The number of matings by males who won fights (within 30 min of mating) was significantly greater than the number of matings by males who were defeated in such fights. Mating rates did not vary significantly with rates of winning at either density. There was a significant power fit between male mating rates and the percentage of fights a male won (irrespective of his fighting-frequency) at the low density. The mean duration a male guarded the female after mating did not vary significantly between densities. There was a significant linear relationship between the duration a spermatophore was retained and the duration a male guarded the female after mating. Courtship song apparently stimulated spermatophore removal. Male guarding involved inter-male aggression and reduced courtship attempts by other males. Males at the high density received no apparent reproductive benefits associated with increased social stimulation. Conclusive evidence for preferential choice of males by females, using the criteria examined here, is lacking. Males at the lower density had fewer competitors and could monopolize females more effectively.

The role of the dopaminergic system in the production of ultrasonic calls in adult rats /

Ultrasonic vocalizations (USV) are emitted by rats in a number of social situations such as aggressive encounters, during sexual behavior, and during play in young rats, situations which are predominantly associated with strong emotional responses. These USV typically involve two distinct types of calls: 22 kHz calls, which are emitted in aversive situations and 50 kHz calls, which are emitted in non-aversive, appetitive situation. The 50 kHz calls are the focus of the present study and to date both the glutamatergic and the dopaminergic systems have been independently implicated in the production of these 50 kHz calls. The present study was conducted to examine a possible relationship between glutamate (GLU) and dopamine (DA) in mediating 50 kHz calls. It was hypothesized that the dopaminergic system plays a mediating role in 50 kHz calls induced by injections ofGLU into the anterior hypothalamic/preoptic area (AHPOA) in adult rats. A total of 68 adult male rats were used in this study. Rats' USV were recorded and analyzed in five experiments that were designed to test the hypothesis: in experiment 1, rats were treated with systemic amphetamine (AMPH) alone; in experiment 2, intra- AHPOA GLU was pretreated with systemic AMPH; in experiment 3, intra-AHPOA GLU was pretreated with intra-AHPOA AMPH; in experiment 4, rats were treated with high and low doses of intra-AHPOA AMPH only; in experiment 5, rats were treated with systemic haloperidol (HAL) as a pretreatment for intra-AHPOA GLU. Analysis of the results indicated that AMPH has a facilitatory effect on 50 kHz USV and that a relationship between DA and GLU in inducing 50 kHz calls does exist. The effect, however, was only observed when DA receptors were antagonized with HAL and was not seen with systemic AMPH pretreatments of intra-AHPOA GLU. The DAGLU relationship at the AHPOA was unclear.

Chronic mild social stress increases neurogenesis in adolescent male rats

Once thought to occur only during specific periods of development, it is now clear that neurogenesis occurs in the rat hippocampus into adulthood. It is wellestablished that stress during adulthood decreases the rate of neurogenesis, but during adolescence, the effects of stress are much less understood. I investigated the effect of short-term or chronic stress during adolescence (daily lhr isolation and change of cage partner from postnatal day (PND) 30-32 or 30-45) on hippocampal neurogenesis. In experiment 1, rats were administered Bromodeoxyuridine (BrdU) daily on PND 30-32, or 46-48, to mark neurogenesis at the beginning of the stressor or after the stressor had ceased, respectively. Neither short-term nor chronic stress had an effect on proliferation or survival (evidenced by BrdU and Doublecortin (Dcx) immunohistochemistry respectively) of cells born at the beginning of the stress procedure. Compared to controls, BrdU-labeling showed chronic stress significantly increased proliferation of cells generated after the stressor had ceased, but survival of new neurons was not supported (Dcx-Iabeling). However, it may be that BrdU injections are inherently stressful. In experiment 2, the stressor (described above) was applied in the absence of BrdU injections. Ki67 (a marker of proliferation) showed that stress transiently increased cell proliferation. Dcx-Iabeling showed that stress also increased neuron survival into adulthood. Labeling with OX.,.42 (a marker of macro phages) suggested that the immune system plays a role in neurogenesis, as stress transiently decreased the number of activated microglia in the hippocampus. It can be concluded that in the adolescent male rat, chronic mild stress increases neurogenesis.

The Influence of Male Diet on Life History Traits of Female Mosquitoes

In the field, mosquitoes characteristically feed on sugars soon after emergence and intermittently during their adult lives. Sugar meals are commonly derived from plant nectar and homopteran honeydew, and without them, adults can only survive for a few days on larval reserves. In addition to sugar, females of most species rely on blood for the initiation and maintenance of egg development; thus their reproductive success depends to some extent on the availability of blood hosts. Males, on the other hand, feed exclusively on sugars. Consequently, their sexual maturation and reproductive success is largely dependent upon access to sugar sources. Plant nectar and homopteran honeydew are the two main sugar sources utilized by mosquitoes in the wild. Previous laboratory studies had shown that differences between nectar sources can affect the survivorship and biting frequency of disease vectoring mosquitoes. However, little is known on how sugar composition influence the reproductive processes in male mosquitoes. Male mosquitoes transfer accessory gland proteins and other hormones to their mates along with sperm during mating. In the female, these seminal fluid constituents exert their influence on reproductive genes that control ovulation and vitellogenesis. The present study tests the hypothesis that the mates of males consuming different sugar meals will exhibit varying levels of induction of vitellogenin (a gene which regulates the expression of egg yolk precursor proteins). Real-time quantitative RT-PCR was used to investigate how each sugar meal indirectly influences vitellogenin mRNA abundance in female Anopheles stephensi following mating. Results indicate that mates of nectar-fed males exhibit 2-fold greater change in vitellogenin expression than the mates of honeydew-fed males. However, this response did not occur in non-blood fed controls. These findings suggest that the stimulatory effect of mating on vitellogenesis in blood meal-reliant (i.e. anautogenous) mosquitoes may only be synergistic in nature. The present study also sought to compare the potential fitness costs of mating incurred by females that do not necessarily require a blood meal to initiate a reproductive cycle (i.e., exhibit autogeny). Females of the facultatively autogenous mosquito, Culex molestus were allowed to mate with males sustained on either nectar or honedyew. Mean lifetime fecundity and survivorship of females under the two different mating regimes were then recorded. Additionally, one-dimensional gel electrophoresis was used to verify the transfer of male accessory gland proteins to the sperm storage organs of females during mating.While there was no significant difference in survival between the test treatments, the mates of nectar-fed males produced 11% more eggs on average than mates of honeydew-fed males. However, additional data are needed to justify the extrapolation of these findings to natural settings. These findings prompt further investigation as the differences caused by diet variation in males may be reflected across other life history traits such as mating frequency and insemination capacity.

Pharmacological analysis of 50 kHz vocalizations in the male rat

The production of 50 kHz ultrasonic vocalizations in rats has been associated with both positive social interactions and appetitive behavioural situations. Furthermore, there is significant evidence showing that these vocalizations are controlled by the meso-limbic dopamine system. The purpose of this study was to perform a pharmacological analysis of 50 kHz calls by using dopamine and two dopamine agonists amphetamine and apomorphine, to induce calls. The acoustic parameters of the different call types were compared across each agonist. All three agonists were able to significantly induce more 50 kHz vocalizations compared to the vehicle control. Furthermore, calls elicited by apomorphine had a significantly higher bandwidth compared to those elicited by dopamine and amphetamine. All three agonists also had significantly different pharmacokinetic properties. These observations suggest that the D2 receptor sub-type is involved in the length of call bandwidths.

Les gènes et protéines BSP chez la souris et l’humain : clonage, caractérisation, expression sous forme recombinante et étude des fonctions biologiques

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins.

Regulation of HMG-CoA reductase, HSL and ACAT expression and activity in testicular cholesterol metabolism in mink and in mouse following experimental genetic deletion

Introduction: L'homéostasie du cholestérol est indispensable à la synthèse de la testostérone dans le tissu interstitiel et la production de gamètes mâles fertiles dans les tubules séminifères. Les facteurs enzymatiques contribuent au maintien de cet équilibre intracellulaire du cholestérol. L'absence d'un ou de plusieurs enzymes telles que la HMG-CoA réductase, la HSL et l'ACAT-1 a été associée à l'infertilité masculine. Toutefois, les facteurs enzymatiques qui contribuent au maintien de l'équilibre intra-tissulaire du cholestérol n'ont pas été étudiés. Cette étude a pour but de tester l'hypothèse que le maintien des taux de cholestérol compatibles avec la spermatogenèse nécessite une coordination de la fonction intracellulaire des enzymes HMG-CoA réductase, ACAT1 et ACAT2 et la HSL. Méthodes: Nous avons analysé l'expression de l’ARNm et de la protéine de ces enzymes dans les fractions enrichies en tubules séminifères (STf) de vison durant le développement postnatal et le cycle reproductif annuel et dans les fractions enrichies en tissu interstitiel (ITf) et de STf durant le développement postnatal chez la souris. Nous avons développé deux nouvelles techniques pour la mesure de l'activité enzymatique de la HMG-CoA réductase et de celle de l'ACAT1 et ACAT2. En outre, l'immunohistochimie a été utilisée pour localiser les enzymes dans le testicule. Enfin, les souris génétiquement déficientes en HSL, en SR-BI et en CD36 ont été utilisées pour élucider la contribution de la HMG-CoA réductase, l'ACAT1 et l'ACAT2 et la HSL à l'homéostasie du cholestérol. Résultats: 1) HMG-CoA réductase: (Vison) La variation du taux d’expression de l’ARNm de la HMG-CoA réductase était corrélée à celle de l'isoforme de 90 kDa de la protéine HMG-CoA réductase durant le développement postnatal et chez l'adulte durant le cycle reproductif saisonnier. L'activité enzymatique de la HMG-CoA réductase augmentait de façon concomitante avec le taux protéinique pour atteindre son niveau le plus élevé à 240 jours (3.6411e-7 mol/min/μg de protéines) au cours du développement et en Février (1.2132e-6 mol/min/μg de protéines) durant le cycle reproductif chez l’adulte. (Souris), Les niveaux d'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase étaient maximales à 42 jours. A l'opposé, le taux protéinique diminuait au cours du développement. 2) HSL: (Vison), l'expression de la protéine de 90 kDa de la HSL était élevée à 180- et 240 jours après la naissance, ainsi qu'en Janvier durant le cycle saisonnier chez l'adulte. L'activité enzymatique de la HSL augmentait durant le développement pour atteindre un pic à 270 jours (36,45 nM/min/μg). Chez l'adulte, l'activité enzymatique de la HSL était maximale en Février. (Souris) Le niveau d’expression de l'ARNm de la HSL augmentait significativement à 21-, 28- et 35 jours après la naissance concomitamment avec le taux d'expression protéinique. L'activité enzymatique de la HSL était maximale à 42 jours suivie d'une baisse significative chez l'adulte. 3) ACAT-1 et ACAT-2: Le présent rapport est le premier à identifier l’expression de l'ACAT-1 et de l'ACAT-2 dans les STf de visons et de souris. (Vison) L'activité enzymatique de l'ACAT-2 était maximale à la complétion du développement à 270 jour (1190.00 CPMB/200 μg de protéines) et en janvier (2643 CPMB/200 μg de protéines) chez l'adulte. En revanche, l'activité enzymatique de l'ACAT-1 piquait à 90 jours et en août respectivement durant le développement et chez l'adulte. (Souris) Les niveaux d'expression de l'ARNm et la protéine de l'ACAT-1 diminuait au cours du développement. Le taux de l'ARNm de l'ACAT-2, à l’opposé du taux protéinique, augmentait au cours du développement. L'activité enzymatique de l'ACAT-1 diminuait au cours du développement tandis que celle de l'ACAT-2 augmentait pour atteindre son niveau maximal à 42 jours. 4) Souris HSL-/ -: Le taux d’expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase diminuaient significativement dans les STf de souris HSL-/- comparés aux souris HSL+/+. Par contre, les taux de l'ARNm et les niveaux des activités enzymatiques de l'ACAT-1 et de l'ACAT-2 étaient significativement plus élevés dans les STf de souris HSL-/- comparés aux souris HSL+/+ 5) Souris SR-BI-/-: L'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase et de l'ACAT-1 étaient plus basses dans les STf de souris SR-BI-/- comparées aux souris SR-BI+/+. A l'opposé, le taux d'expression de l'ARNm et l'activité enzymatique de la HSL étaient augmentées chez les souris SR-BI-/- comparées aux souris SR-BI+/+. 6) Souris CD36-/-: L'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase et de l'ACAT-2 étaient significativement plus faibles tandis que celles de la HSL et de l'ACAT-1 étaient inchangées dans les STf de souris CD36-/- comparées aux souris CD36+/+. Conclusion: Nos résultats suggèrent que: 1) L'activité enzymatique de la HMG-CoA réductase et de la HSL sont associées à l'activité spermatogénétique et que ces activités ne seraient pas régulées au niveau transcriptionnel. 2) L'ACAT-1 et de l'ACAT-2 sont exprimées dans des cellules différentes au sein des tubules séminifères, suggérant des fonctions distinctes pour ces deux isoformes: l'estérification du cholestérol libre dans les cellules germinales pour l'ACAT-1 et l'efflux du cholestérol en excès dans les cellules de Sertoli au cours de la spermatogenèse pour l'ACAT-2. 3) La suppression génétique de la HSL diminuait la HMG-CoA réductase et augmentait les deux isoformes de l'ACAT, suggérant que ces enzymes jouent un rôle critique dans le métabolisme du cholestérol intratubulaire. 4) La suppression génétique des transporteurs sélectifs de cholestérol SR-BI et CD36 affecte l'expression (ARNm et protéine) et l'activité des enzymes HMG-CoA réductase, HSL, ACAT-1 et ACAT-2, suggérant l'existence d’un effet compensatoire entre facteurs enzymatiques et non-enzymatiques du métabolisme du cholestérol dans les fractions tubulaires. Ensemble, les résultats de notre étude suggèrent que les enzymes impliquées dans la régulation du cholestérol intratubulaire agissent de concert avec les transporteurs sélectifs de cholestérol dans le but de maintenir l'homéostasie du cholestérol intra-tissulaire du testicule.