Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

<p>Abstract</p> <p>Background</p> <p>Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.</p> <p>Methods</p> <p>Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.</p> <p>Results</p> <p>The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).</p> <p>Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.</p> <p>Conclusion</p> <p>Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.</p

Passive immune transfer in puppies

Le chiot nait presque agammaglobulinémique. Il acquiert une immunité passive systémique grâce au colostrum au cours des deux premiers jours de vie, La qualité du transfert d’immunité passive (appréciée par la concentration circulante des IgG à deux jours de vie) a un impact sur la santé du chiot et sur son taux de mortalité (multiplié par neuf en cas de déficit de transfert) mais interfère avec l’efficacité vaccinale. Elle est très variable entre portées ainsi qu’entre les chiots d’une même portée. La concentration des IgG du colostrum semble avoir peu d’impact sur la qualité du transfert de l’immunité passive. Ce transfert dépend davantage du délai écoulé entre la naissance et l’ingestion du colostrum du fait, du côté maternel, de la détérioration rapide de la qualité immunologique du colostrum (qui chute de plus de 50% au cours des 24 premières heures post partum) et du côté du nouveau-né, de la fermeture de la barrière intestinale (la perméabilité de l’intestin du chiot aux IgG diminue de moitié toutes les quatre heures pour devenir nulle au-delà de 12 heures de vie). L’activité sérique des gammaglutamyltranférases permet le diagnostic du déficit de transfert d’immunité passive (sensibilité : 87,5% ; spécificité : 80%). Ce déficit peut également être diagnostiqué par le calcul du taux de croissance entre la naissance et l’âge de deux jours (sensibilité : 96,3% ; spécificité : 83,1%). En l’absence de colostrum, peu de solutions sont disponibles pour faire acquérir un transfert d’immunité adéquat : la constitution d’une banque de colostrum est la solution optimale. Outre le transfert d’immunité systémique, les anticorps maternels (principalement les IgA) assurent une immunité locale, digestive dont les rôles à moyen terme pour la protection du chiot contre les entéropathogènes et, à long terme dans l’éduction du système immunitaire digestif, restent à explorer.The puppy, born without immunoglobulins G (IgG), acquires a passive systemic immunity thanks to colostrum during the two first days of life. The quality of passive immune transfer (i.e. blood IgG concentration at two days of age) impacts puppy’s health and its mortality rate but interferes with response to vaccination. It is highly variable between litters and between puppies within litters. Colostrum IgG concentration is of very limited influence on passive immune transfer, which rather depends on the time elapsed between birth and ingestion of colostrum. Deficit in passive immune transfer can be diagnosed through blood gammaglutamyltranferases assay and growth rate over the two first days of life. Colostrum banking is the optimal solution for orphan puppies. In addition to systemic passive immune transfer, maternal antibodies (mainly IgA) would provide local (digestive) immunity, ensuring mid-term protection of the puppies gut together with probably long term training of the digestive immune system

In vitro maturation of canine oocyte

Compared to other mammals, the canine oocyte offers a very unusual model of meiosis. Its maturation in vitro, studied only over the past ten years, is still poorly controlled: low rate of metaphase II (10 to 20% vs. over 90% in cattle), and high rate of degeneration in cultures (20 to 60%) despite attempts to improve culture media. However, in dogs as well as in canidae threatened by extinction, in vitro maturation is a key step for reproductive biotechnologies, such as in vitro fertilization and embryo production. It is therefore urgent to improve our understanding of the canine oocyte to improve maturation rates. We initiated studies on oocyte maturation in bitches. We examined the role of cAMP in the resumption of meiosis in vitro in bitches, using substances which reduce (Rp-cAMP) or increase (dbcAMP and forskolin) its level inside the oocyte. We also used denuded oocytes to prevent any cAMP supply from the granulosa cells. With this model, we showed that cAMP might play a role in maintaining meiosis, and that the resumption of meiosis may also be controlled by another pathway, possibly involving calcium. Another of our research projects explores changes in the oocyte ultrastructure during in vivo and in vitro maturation. Transmission electron microscopy may provide precise information on possible cytoplasmic anomalies induced by maturation. This fundamental work will eventually help us improve in vitro maturation of canine oocytes.L'ovocyte de chienne constitue un modèle de méiose très particulier parmi les mammifères. Sa maturation in vitro, étudiée depuis une dizaine d'années seulement, reste très mal maîtrisée : faible taux de métaphase II (10 à 20 % contre plus de 90 % chez les bovins) et fort taux de dégénérescence en culture (20 à 60 %) malgré les essais d'amélioration des milieux de culture. Or la maturation in vitro est une étape indispensable pour avoir accès, tant chez le chien que chez les Canidés en voie de disparition, aux biotechnologies de la reproduction (fécondation et production d'embryons in vitro notamment). Il est indispensable de mieux comprendre la biologie de l'ovocyte chez la chienne pour améliorer les taux de maturation. Nous nous sommes intéressées, en premier lieu, au rôle de l'AMPc dans la reprise de la méiose in vitro. Nous avons modulé la concentration intraovocytaire d'AMPc en soumettant les ovocytes à des molécules qui la diminuent (Rp-AMPc) ou l'augmentent (dbAMPc et forskoline), ou en dénudant l'ovocyte pour arrêter tout apport par les cellules du cumulus. Nous avons ainsi montré que l'AMPc jouerait un rôle dans la poursuite de la méiose dont la reprise serait également contrôlée par une autre voie, peut-être calcique. En parallèle, nous explorons l'évolution de l'ultrastructure de l'ovocyte au cours de la maturation in vivo et in vitro, pour détecter les anomalies cytoplasmiques qui peuvent apparaître au cours de la maturation

Polyovular ovarian follicles: physiological and not so rare

In mammalian ovaries, the vast majority of follicles contain only one oocyte. However, follicles containing several oocytes, or polyovular follicles, are also found in most species. Their frequency ranges from < 0.1% to 14% of the total number of follicles, and they can contain from 2 to 17 oocytes (or even 100 in certain marsupials). Three hypotheses have been proposed to explain their occurrence: division of an oocyte initially containing several nuclei, fusion of several different follicles, or nonseparation of several oocytes at the time of the formation of the primordial follicles. This latter hypothesis seems the most likely. The fate of these follicles has not been studied extensively, and for a long time they were considered as pathological. Studies conducted in pigs and dogs have shown that these polyovular follicles can grow, reach the preovulatory stage, and ovulate. However, all the oocytes in a single follicle are not equal. The examination of oocytes retrieved from a single follicle suggests that only one oocyte shows good morphological characteristics, whereas the others are at various stages of more or less advanced degeneration. The regulation of the number of polyovular follicles is still poorly understood and the data on the effects of age, gonadotropins, and steroids is contradictory. Their frequency can be increased by using treatments with estrogenic effects, or endocrine disruptors. In mice, the control is also genetic. The mechanisms leading to the formation of polyovular follicles remain to be explored, and the study of the functioning of these particular follicles would improve our understanding of the oocyte-follicle dialogue.Dans les ovaires des mammifères, la très grande majorité des follicules ne contient qu'un seul ovocyte. Mais, dans la plupart des espèces, certains follicules contiennent plusieurs ovocytes: on parle alors de follicules polyovocytaires ou multiovocytaires. Leur fréquence varie entre <0.1 % et 14% des follicules totaux et ils peuvent contenir de 2 à 17 ovocytes (et même plus de 100 chez certains marsupiaux). Trois hypothèses pourraient expliquer leur formation: la division d'un ovocyte ayant initialement plusieurs noyaux, la fusion de plusieurs follicules différents ou la non-séparation de plusieurs ovocytes au moment de la formation des follicules primordiaux. Cette dernière hypothèse semble la plus probable. La destinée de ces follicules a été encore peu explorée et ils ont été longtemps considérés comme pathologiques. Des travaux menés notamment chez le porc et le chien montrent que ces follicules polyovocytaires peuvent grandir, atteindre le stade préovulatoire et ovuler. Cependant, tous les ovocytes d'un même follicule ne sont pas équivalents. L'observation des ovocytes après ponction du follicule suggère qu'un seul ovocyte semble de bonne qualité morphologique, tandis que les autres sont à des stades de dégénérescence plus ou moins avancés. La régulation du nombre des follicules polyovocytaires est peu connue et il existe des données contradictoires sur les effets de l'âge, des gonadotrophines et des stéroïdes. Leur fréquence peut être augmentée par des traitements à effets œstrogéniques ou par l'action de perturbateurs endocriniens. Chez la souris, le contrôle est également génétique. Les mécanismes conduisant à la formation des follicules polyovocytaires restent à explorer et l'étude du fonctionnement de ces follicules pourrait contribuer à une meilleure connaissance du dialogue ovocyte-follicule

Characterization of oviduct epithelial spheroids for the study of embryo-maternal communication in cattle

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo-maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100-200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo-maternal interactions in cattle

Canine oocyte maturation, fertilization and early embryonic development

Canine reproduction has several distinctive features. Firstly, folliculogenesis is unusual as numerous ovarian follicles contain several oocytes (polyovular follicles). Secondly, unlike in other mammalian species, oocytes at the time of ovulation are still at an immature stage (prophase I, germinal vesicle stage), and complete their maturation in the oviduct. This phenomenon is not easy to observe because the canine oocyte has a high lipid content and its DNA is difficult to visualise. Fertilization of immature oocytes has been observed in vitro, however in vivo, fertilization occurs in oocytes at the metaphase II stage, approximately 50 hours after ovulation. The 2-pronuclei stage is reached 72-124 hours after ovulation, and 2-cell embryos are present 96-168 hours after ovulation. The oviductal phase is long and embryos enter the uterine cavity at the morula or early blastocyst stage 10-12 days following ovulation. Implantation occurs 18 to 21 days after ovulation. In spite of all these specificities, studies on canine reproduction were so far mainly clinical. However, current research is focusing on fundamental knowledge, namely the mechanisms controlling oocyte maturation in vivo, in the hope to improve the yield of oocyte maturation in vitro, which is still very low.Plusieurs aspects de la reproduction sont particuliers à l'espèce canine. D'une part, la folliculogenèse est singulière car chez la chienne, de nombreux follicules ovariens contiennent plusieurs ovocytes (follicules poly-ovocytaires). D'autre part, contrairement à ce qui est observé chez les autres femelles de mammifères, au moment de l'ovulation, l'ovocyte est encore à un stade immature (prophase I, stade vésicule germinative ou VG) et la maturation ovocytaire se poursuit ensuite dans l'oviducte. L'observation de ce phénomène est rendue complexe par le fait que l'ovocyte canin est riche en lipides et que son ADN est donc difficile à visualiser. In vitro, la fécondation d'ovocytes immatures a été observée mais in vivo, elle a lieu au moment où les ovocytes ont atteint le stade métaphase II, environ 50 h après l'ovulation. Les premiers pronoyaux sont présents 72 à 124 h après l'ovulation et les premiers embryons au stade 2-cellules sont observés 96 à 168 h après l'ovulation. La période de transit dans l'oviducte est longue et les embryons n'atteignent l'utérus qu'au stade morula ou jeune blastocyste, 10 à 12 jours après l'ovulation et l'implantation a ensuite lieu vers 18 à 21 jours. Globalement, malgré toutes ces particularités, les recherches sur la reproduction dans l'espèce canine étaient jusqu'alors essentiellement cliniques. Les travaux visent maintenant à améliorer les connaissances fondamentales, notamment concernant les mécanismes contrôlant la maturation ovocytaire in vivo, pour pouvoir ensuite améliorer les rendements de la maturation in vitro, actuellement très faibles

Oviductal microenvironment: role in canine oocyte maturation in vivo and in vitro

In most mammals, oocytes are ovulated at the metaphase II stage, and the meiosis inhibition is then lifted by fertilization. In bitches and other Canidae species however, oocytes are released at the prophase I stage, and another 48 to 72h are necessary for themtomature into themetaphase II stage and become fertilizable. This specificity is currently hindering the development of reproductive biotechnologies in these species. In vitro maturation rates of canine oocytes are very low, as only 10 to 30%will reach the metaphase stage after 72h in culture. In bitches, nuclear maturation occurs in the oviduct, and tubal derivatives (culture media, such as Synthetic Oviductal Fluid, oviductal explants, coculture on tubal cell layers) were used to improve the yield, but so far not very successfully. This failure may be due to the lack of data on the composition of oviductal fluid in bitches. Further studies on the oviductal microenvironment of bitches are therefore necessary, as it is probably quite different from the oviductalmicroenvironment of other females, e.g. the presence of preovulatory luteinisation in bitches only. Creating a maturation medium based on the composition of oviductal fluid could be an interesting avenue to explore to improve in vitro maturation rates.Chez la plupart des mammifères, les ovocytes sont bloqués en métaphase II au moment de l'ovulation et cette inhibition de la méiose est ensuite levée par la fécondation. Chez les chiennes et les autres femelles de canidés, les ovocytes sont libérés au stade de prophase I, et il faut encore attendre 48 à 72 heures pour qu'ils atteignent le stade de métaphase II et deviennent fécondables. Cette particularité constitue aujourd'hui un frein au développement des biotechnologies de la reproduction chez les canidés. En effet, dans les essais de maturation in vitro d'ovocytes canins, seuls 10 à 30 % des ovocytes atteignent le stade de métaphase au bout de 72 heures de culture. Chez la chienne, la maturation nucléaire se produisant dans l'oviducte, des substituts de l'oviducte (milieux de culture comme le Synthetic Oviductal Fluid, explants d'oviductes, cultures sur tapis de cellules tubaires) ont été utilisés pour les cultures d'ovocytes in vitro, afin d'en améliorer le rendement, mais sans grand succès jusqu'à maintenant. Cet échec peut être dû au manque de données sur la composition du liquide tubaire de la chienne. L'étude de ce microenvironnement prend donc tout son intérêt, celui-ci étant probablement assez différent de celui des autres femelles, ne serait-ce que par l'existence du processus de lutéinisation préovulatoire dans cette espèce. À terme, la conception d'un milieu de maturation sur la base de la composition du liquide tubaire pourrait être une voie intéressante pour augmenter les taux de maturation in vitro

Embryo biotechnologies in dogs

There is very little data available on the specificities of oocyte and embryo biology in bitches. The main difference with other mammals is the time of meiosis resumption: it does not occur before ovulation, but in the oviduct 48 to 60 hours later. The factors responsible for this delay are not known, which may explain why current in vitro maturation rates are so low (10 to 30%). Oocyte harvesting is also a major limiting factor, as there is no effective protocol for the induction of cycles and superovulation. In vitro fertilisation rates are equally low (10%), with a high rate of polyspermia. No puppy has yet been born from an embryo produced in vitro. As for embryos produced in vivo, their collection is difficult due to anatomical reasons and to the fact that superovulation cannot be induced. Embryo transfer to donor bitches is also hindered by difficulties to synchronise ovulations between donor and recipient bitches. Only 6 such trials have been reported in the literature, resulting in the birth of 45 puppies. In vitro cultures are very rarely used, and only four puppies were born from somatic cell cloning with only few hours of in vitro culture. Canine reproductive biotechnologies have thus largely fallen behind, due to a lack of fundamental research to improve our understanding of its specific physiological mechanisms. This deficit is all the more damaging that dogs are increasingly used as relevant biomedical models.La biologie de l'ovocyte et de l'embryon canins, qui présentent des particularités spécifiques, est très mal connue. La chienne se distingue principalement par les modalités de reprise de la méiose ovocytaire : celle-ci n'a pas lieu au moment de l'ovulation, comme chez les autres femelles mammifères, mais dans l'oviducte 48 à 60 heures après l'ovulation. Les facteurs responsables de ce retard ne sont pas connus. Ceci explique sans doute pourquoi les taux de maturation in vitro obtenus à l'heure actuelle sont faibles (10 à 30 %). La collecte des ovocytes est aussi un facteur limitant majeur, en l'absence de protocole efficace d'induction des cycles et de superovulation. Les rendements de fécondation in vitro sont également faibles (10 %), avec un taux élevé de polyspermie. À l'heure actuelle, aucun chiot n'est encore né à partir d'un embryon produit in vitro. Quant aux embryons produits in vivo, leur collecte est difficile pour des raisons anatomiques et le rendement est limité par l'impossibilité d'induire des superovulations; par ailleurs, leur transfert chez des femelles receveuses se heurte aux difficultés de synchronisation des ovulations entre la femelle donneuse et les receveuses, et la littérature ne décrit que six essais, qui ont abouti à la naissance de 45 chiots. Avec un très faible recours à la culture in vitro, quatre naissances de chiots ont été obtenues par clonage de cellules somatiques. Les biotechnologies de la reproduction sont donc largement en retard dans l'espèce canine, qui souffre d'un manque de travaux fondamentaux visant à mieux comprendre ses mécanismes physiologiques spécifiques. Ce déficit est d'autant plus dommageable que le chien prend une place croissante et pertinente en tant que modèle biomédical

Development of a Multivariate Prediction Model for Early-Onset Bronchiolitis Obliterans Syndrome and Restrictive Allograft Syndrome in Lung Transplantation.

Chronic lung allograft dysfunction and its main phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), are major causes of mortality after lung transplantation (LT). RAS and early-onset BOS, developing within 3 years after LT, are associated with particularly inferior clinical outcomes. Prediction models for early-onset BOS and RAS have not been previously described. LT recipients of the French and Swiss transplant cohorts were eligible for inclusion in the SysCLAD cohort if they were alive with at least 2 years of follow-up but less than 3 years, or if they died or were retransplanted at any time less than 3 years. These patients were assessed for early-onset BOS, RAS, or stable allograft function by an adjudication committee. Baseline characteristics, data on surgery, immunosuppression, and year-1 follow-up were collected. Prediction models for BOS and RAS were developed using multivariate logistic regression and multivariate multinomial analysis. Among patients fulfilling the eligibility criteria, we identified 149 stable, 51 BOS, and 30 RAS subjects. The best prediction model for early-onset BOS and RAS included the underlying diagnosis, induction treatment, immunosuppression, and year-1 class II donor-specific antibodies (DSAs). Within this model, class II DSAs were associated with BOS and RAS, whereas pre-LT diagnoses of interstitial lung disease and chronic obstructive pulmonary disease were associated with RAS. Although these findings need further validation, results indicate that specific baseline and year-1 parameters may serve as predictors of BOS or RAS by 3 years post-LT. Their identification may allow intervention or guide risk stratification, aiming for an individualized patient management approach