Search

Investigating the roles of chemical factors stimulating and inhibiting sperm motility is required to understand the mechanisms of spermatozoa movement. In this study, we described the composition of the seminal fluid (osmotic pressure, pH, and ions) and investigated the roles of these factors and salinity in initiating spermatozoa movement in the Pacific oyster, Crassostrea gigas. The acidic pH of the gonad (5.82±0.22) maintained sperm in the quiescent stage and initiation of flagellar movement was triggered by a sudden increase of spermatozoa external pH (pHe) when released in seawater (SW). At pH 6.4, percentage of motile spermatozoa was three times higher when they were activated in SW containing 30 mM NH4Cl, which alkalinizes internal pH (pHi) of spermatozoa, compared to NH4Cl-free SW, revealing the role of pHi in triggering sperm movement. Percentage of motile spermatozoa activated in Na+-free artificial seawater (ASW) was highly reduced compared to ASW, suggesting that change of pHi triggering sperm motility was mediated by a Na+/H+ exchanger. Motility and swimming speed were highest in salinities between 33.8 and 42.7‰ (within a range of 0 to 50 ‰), and pH values above 7.5 (within a range of 4.5 to 9.5)., Summary: Oyster sperm motility is not triggered in an acidic environment and motility is controlled by internal pH alkalinization, mediated by a Na+/H+ exchanger.

Pacific oyster hatchery production remains highly variable, partially due to inconsistent gamete quality. However, little attention has been paid to oocyte quality in Pacific oyster compared to important vertebrate aqua-culture species such as fishes. The aims of the present study were to identify criteria of oocyte and trochophore quality used as predictive tools for embryo developmental success at the D-larval stage. Twenty-six potential proxies of oocyte and trochophore quality were studied on 25 mature females, as determined using histology. Some morphological and biochemical characteristics of mature oocytes were described. Protein appeared to be the main constituent of fully mature Pacific oyster oocytes. Neutral lipids accounted for 61% of the total lipids and triglycerides for 85% of the neutral lipids. Oocyte fertilization capacity was assessed by determining the D-larval yield. High inter-individual variation of D-larval yield was observed (ranging from 1% to 58%). D-larval yield was positively correlated with the percentage of motile trochophores (R-2 = 0.237) and with trochophore velocity of the average path (VAP; R-2 = 0.255) and negatively correlated with oocyte total steryl esters (R-2 = 0.244). The use of a multivariate model, including D-larval yield, trochophore VAP and total steryl esters, led to a higher coefficient of determination (R-2 = 0.352), highlighting that the prediction of embryo development in mollusks is a complex process requiring sophisticated multi-factor models.

In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied 1) changes in spermatozoal mitochondrial activity, that is, mitochondrial membrane potential (MMP), and intracellular ATP content in relation to motion parameters and 2) the involvement of OXPHOS for spermatozoal movement using carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoal ATP content decreased steadily during the first 9 h postactivation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that the ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or from potential phosphagens during the first 2 h of movement as deduced by the decoupling by CCCP of OXPHOS. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyze intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.

The present study aims at describing the energy metabolism of Pacific oyster, Crassostrea gigas, spermatozoon. The main objectives are: (1) describing the kinetic of cellular and biochemical characteristics of Pacific oyster spermatozoon during its movement phase, (2) identifying the metabolic pathways to produce energy and their substrates, (3) determining the involvement of energy metabolism in fertilization success. The results showed the long duration (ranging 24 to 72h) of the movement of Pacific oyster spermatozoon and the maintenance of a high intracellular ATP content until the end of the movement phase. The role of oxidative phosphorylation (OXPHOS) in ATP production was highlighted. Alternative ATP producing pathways might contribute to sustain energy production when OXPHOS is impaired, showing the high adaptation capacity of Pacific oyster spermatozoa. An increase of phosphoarginine content observed at the end of the movement phase suggests energy storage capacity. Endogenous substrates involved in ATP synthesis must be determined. Both the high regulation capacity of the energy metabolism of Pacific oyster spermatozoa and their ability to restore energy stores catabolized during movement likely contribute to the successful settlement observed in the wild. In conclusion, this work provided a fine description of energetic metabolism of Pacific oyster spermatozoon., Cette thèse présente une étude du métabolisme énergétique du spermatozoïde d’huître creuse, Crassostrea gigas. Les principaux objectifs sont (1) d’établir la dynamique des caractéristiques cellulaires et biochimiques du spermatozoïde de cette espèce durant la phase de mouvement, (2) d’identifier les voies de synthèse d’ATP et leurs substrats et (3) de déterminer l’implication du métabolisme énergétique dans le succès à la fécondation du spermatozoïde de cette espèce. Les résultats de ce travail montrent la longue durée du mouvement (de 24 à 72h) du spermatozoïde d’huître creuse et le maintien d’une concentration élevée en ATP intracellulaire, jusqu'à la fin du mouvement. Le rôle de la phosphorylation oxydative (OXPHOS) dans la synthèse d’ATP a été mis en évidence. Le spermatozoïde d’huître creuse montre une grande plasticité dans l’utilisation de ses voies de production d’énergie, assurant la synthèse d’ATP par d’autres voies qu’OXPHOS lorsque la mitochondrie est inhibée. L’accumulation de phosphoarginine en fin de mouvement montre une capacité de stockage de l’ATP synthétisé. La nature des substrats endogènes utilisés pour la synthèse d’ATP reste cependant à déterminer. La forte capacité de régulation du métabolisme énergétique du spermatozoïde d’huître creuse, associée à la possibilité de synthétiser de l’énergie au cours de sa longue phase de nage, contribuent probablement au succès du peuplement observé chez cette espèce. Ce travail apporte une première description du métabolisme énergétique du spermatozoïde d’huître creuse, améliorant la connaissance fine de cette cellule.

The present study aims at describing the energy metabolism of Pacific oyster, Crassostrea gigas, spermatozoon. The main objectives are: (1) describing the kinetic of cellular and biochemical characteristics of Pacific oyster spermatozoon during its movement phase, (2) identifying the metabolic pathways to produce energy and their substrates, (3) determining the involvement of energy metabolism in fertilization success. The results showed the long duration (ranging 24 to 72h) of the movement of Pacific oyster spermatozoon and the maintenance of a high intracellular ATP content until the end of the movement phase. The role of oxidative phosphorylation (OXPHOS) in ATP production was highlighted. Alternative ATP producing pathways might contribute to sustain energy production when OXPHOS is impaired, showing the high adaptation capacity of Pacific oyster spermatozoa. An increase of phosphoarginine content observed at the end of the movement phase suggests energy storage capacity. Endogenous substrates involved in ATP synthesis must be determined. Both the high regulation capacity of the energy metabolism of Pacific oyster spermatozoa and their ability to restore energy stores catabolized during movement likely contribute to the successful settlement observed in the wild. In conclusion, this work provided a fine description of energetic metabolism of Pacific oyster spermatozoon., Cette thèse présente une étude du métabolisme énergétique du spermatozoïde d’huître creuse, Crassostrea gigas. Les principaux objectifs sont (1) d’établir la dynamique des caractéristiques cellulaires et biochimiques du spermatozoïde de cette espèce durant la phase de mouvement, (2) d’identifier les voies de synthèse d’ATP et leurs substrats et (3) de déterminer l’implication du métabolisme énergétique dans le succès à la fécondation du spermatozoïde de cette espèce. Les résultats de ce travail montrent la longue durée du mouvement (de 24 à 72h) du spermatozoïde d’huître creuse et le maintien d’une concentration élevée en ATP intracellulaire, jusqu'à la fin du mouvement. Le rôle de la phosphorylation oxydative (OXPHOS) dans la synthèse d’ATP a été mis en évidence. Le spermatozoïde d’huître creuse montre une grande plasticité dans l’utilisation de ses voies de production d’énergie, assurant la synthèse d’ATP par d’autres voies qu’OXPHOS lorsque la mitochondrie est inhibée. L’accumulation de phosphoarginine en fin de mouvement montre une capacité de stockage de l’ATP synthétisé. La nature des substrats endogènes utilisés pour la synthèse d’ATP reste cependant à déterminer. La forte capacité de régulation du métabolisme énergétique du spermatozoïde d’huître creuse, associée à la possibilité de synthétiser de l’énergie au cours de sa longue phase de nage, contribuent probablement au succès du peuplement observé chez cette espèce. Ce travail apporte une première description du métabolisme énergétique du spermatozoïde d’huître creuse, améliorant la connaissance fine de cette cellule.

International audience; The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques.

International audience

The present chapter aims at describing the main biological characteristics of two oyster species, both important for their commercial values. Oyster spermatozoa belong to the "aquasperm" according to ultra-structural data: they possess an acrosome and four mitochondria. The sperm flagellum is devoid of progressive motility eitheir in the gonads or at spawning in sea water. In both cases, flagellar motility (% of moving cells) appears slowly in a time dependent manner in natural sea water (5 to 15 min). This ability to acquire potentiality for motility (maturation process) can be accelerated by sperm incubation with drugs like serotonine, theophilline, caffeine or dibutyryl-cAMP, which rapidly initiate progressive movement of live sperm. All drugs are effective in a time dependent manner and especially caffeine and theophilline are shown to act in a concentration dependent manner. These drugs allow forwardly directed translation for the majority of sperm cells. Furtheremore, spermatozoa induced to such efficient swimming behavior exhibit fertilizing performances of high levelDuring hatchery practices, oyster sperm are frequently collected by scarification of the male gonad ; alternatively, ejaculation can be induced by various methods. According to our results, injection of compounds such as serotonine (1 to 10mM) in the male gonad leads to release of sperm in sea water. By dilution into regular sea water, live oyster spermatozoa obtained by scarification show initially mostly erratic movements with poor efficiency for forward translation and initiate efficient flagellar motility after several minutes.Motility of black-lip pearl oyster spermatozoa appears gradually when spermatozoa are transferred into alkaline sea water. This motility-activating effect is reversed when pH is shifted back to acidic values; as in normal sea water, activity of sperm (% motile cells) increases gradually after alkaline pH activation and lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is ranging 230 μm/sec. The swimming tracks are almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural sea water at 4°C retain potent motility for several days and can be subsequently activated by alkaline sea water.In case of the Pacific oyster, motility appears also to be under the control of acidic pH. This would explain how motility activation occurs when inactive sperm in seminal fluid (pH 5.5) comes in contact with sea water (pH 8.0) at shedding. Sperm movement is hasting for a long period (Pacific oyster: 20 to 72 h), suggesting spermatozoa could restore their energy stores. Therefore, the cessation of Pacific oyster sperm movement is unlikely caused by an insufficient concentation of ATPThis book chapter intends to describe in both species details of the present knowledge on the characteristics of spermatozoa, including their swimming behaviour in relation with their energetic content (ATP) and their ability to be sustained by mitochondrial respiration, in a comparative way with other marine animals.

International audience