Joëlle Marchandise

Cell Cycle Duration at the Time of Maternal Zygotic Transition for In Vitro Produced Bovine Embryos: Effect of Oxygen Tension and Transcription Inhibition

Abstract Early embryonic cleavages are mostly regulated by maternal components then control of development progressively depends on newly synthesized zygotic products. The timing of the first cleavages is a way to assess embryo quality. The goal of this study was to evaluate the duration of the fourth cell cycle, at the time of maternal-to-zygotic transition (MZT) in in vitro-produced bovine embryos by means of cinematographic analysis. We found that 75% of the embryos displayed a long fourth cycle (43.5 ± 5.4 h) whereas the remaining embryos had a very short fourth cell cycle (8.9 ± 2.9 h). Both groups did not differ in cleavage rhythm up to the eight-cell stage and timing of cavitation and blastocyst expansion was identical. However, embryos with a short fourth cell cycle had a better blastocyst rate than embryos with a long cycle (59% versus 38%, P < 0.01). Total cell number, inner cell mass (ICM):total cell ratio, and hatching rate were identical for blastocysts produced from embryos with either a long or a short fourth cell cycle. In a second experiment, we showed that increasing the oxygen tension, from 5% to 20%, decreased the percentage of embryos with a short fourth cell cycle, from 25% to 11% (P < 0.01), indicating that suboptimal culture conditions can influence the length of this cycle. Finally, we investigated whether fourth cell cycle duration could be influenced by transcription inhibition. With alpha-amanitin added at 18 h postinsemination (HPI), cleavage was reduced (66% versus 79%) and, at 70 HPI, the 9- to 16-cell rate increased (50% versus 25%) concomitantly with a 5- to 8-cell rate decrease (16% versus 47%). A similar pattern was observed when the drug was added at 6 HPI or 42 HPI but not at 0 HPI. Cinematographic analysis revealed that alpha-amanitin increased the first cell cycle duration whereas the second and third cell cycles were not affected. With the drug, one third of the embryos could develop up to the 9- to 16-cell stage and they all had a short fourth cell cycle (11.2 ± 3.7 h) with a good synchrony of cleavage between blastomeres. These results suggest that duration of the fourth cell cycle of bovine embryo, during the MZT, is under a zygotic transcriptional control that can be affected by oxidative conditions.

Poly(A) RNA Is Reduced by Half During Bovine Oocyte Maturation but Increases when Meiotic Arrest Is Maintained with CDK Inhibitors

Abstract Variations in the amount of different RNA species were investigated during in vitro maturation of bovine oocytes. Total RNA content was estimated to be 2 ng before meiosis, and after meiosis resumption, no decrease was observed. Ribosomal RNA did not appear to be degraded either, whereas poly(A) RNA was reduced by half after meiosis resumption, from 53 pg to 25 pg per oocyte. Real-time polymerase chain reaction was performed on growth and differentiation factor-9 (GDF-9), on cyclin B1, and on two genes implicated in the resistance to oxidative stress, glucose-6-phosphate-dehydrogenase (G6PD) and peroxiredoxin-6 (PRDX6). When these transcripts were reverse-transcribed with hexamers, the amplification results were not different before or after in vitro maturation. But when reverse transcription was performed with oligo(dT), amplification was dramatically reduced after maturation, except for cyclin B1 mRNA, implying deadenylation without degradation of three transcripts. Although calf oocytes have a lower developmental competence, their poly(A) RNA contents were not different from that of cow oocytes, nor were they differently affected during maturation. When bovine oocytes were maintained in vitro under meiotic arrest with CDK inhibitors, their poly(A) RNA amount increased, but this rise did not change the poly(A) RNA level once maturation was achieved. The increase could not be observed under transcription inhibition and, when impeding transcription and adenylation, the poly(A) RNA decreased to a level normally observed after maturation, in spite of the maintenance of meiotic arrest. These results demonstrate the importance of adenylation and deadenylation processes during in vitro maturation of bovine oocytes.

Impact of adding 5.5 mM glucose to SOF medium on the development, metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.

Endoplasmic reticulum accumulation of Kir6.2 without activation of ER stress response in islet cells from adult Sur1 knockout mice

Trafficking of pancreatic KATP channels to the plasma membrane critically depends on masking the endoplasmic reticulum (ER) retention signals of the SUR1 and Kir6.2 subunits upon their proper assembly into functional hetero-octamers. When expressed in the absence of the partner protein, each subunit might accumulate in the ER and trigger β-cell ER stress and oxidative stress. To test this hypothesis, Kir6.2 localisation, ER ultra-structure and ER-stress- and oxidative-stress-response gene mRNA levels were evaluated in pancreatic endocrine cells from adult wild-type (WT) and Sur1 knockout (Sur1-/-) mice. As previously reported, Kir6.2 was mainly expressed on secretory granules and at the plasma membrane of WT islet cells. In contrast, like the ER chaperone calreticulin, Kir6.2 was primarily localised in the rough endoplasmic reticulum (RER) of Sur1-/- islet cells. ER retention of Kir6.2 was demonstrated (electron microscopy) by a significant increase in the length and Kir6.2 density of RER in Sur1-/- vs WT islet cells. Despite Kir6.2 retention in RER, Xbp1 mRNA splicing and mRNA levels of preproinsulin and ER-stress-response genes Bip, Edem and Gadd153 were similar in WT and Sur1-/- islets. However, mRNA levels of the antioxidant enzymes Sod1, Sod2, Gpx2 and catalase were significantly up-regulated in Sur1-/- islets. Sequestration of Kir6.2 in RER of Sur1-/- islet cells is thus associated with an increase in RER length and mild oxidative stress without activation of the classical ER stress response.

Impact of Sur1 gene inactivation on the morphology of mouse pancreatic endocrine tissue

In congenital hyperinsulinism of infancy (CHI), the loss of K-ATP channels (composed of Kir6.2 and SUR1 subunits) in β cells induces permanent insulin secretion and severe hypoglycaemia. By contrast, Sur1−/− mice do not present such defects. We have investigated the impact of Sur1 gene inactivation on mouse islet cell morphology, structure and basic physiology. Pancreata were collected from young, adult and old wild-type (WT) and Sur1−/− mice. After immunostaining for hormone, the total endocrine tissue, cell proportion, cell size and intra-insular distribution, hormone content and Glut-2 expression were quantified by morphometry. Basic physiological parameters were also measured. In young Sur1−/− mice, the total endocrine tissue and proportion of β cells were higher (P<0.05) than in WT mice, whereas the proportion of δ cells was lower (P<0.01). In old Sur1−/− mice, α cells were frequently located in the central regions of islets (unlike WT islets) and their proportion was increased (P<0.05). Glut-2 protein and mRNA levels were lower in old Sur1−/− islets (P<0.02). Insulinaemia, fasting insulin and glucagon contents were equivalent in both groups of pancreata. Thus, the islets of Sur1−/− mice present morphological modifications that have not been described in CHI and that might reflect an adaptive mechanism controlling insulin secretion in these mice.

Characterization of β-cell plasticity mechanisms induced in mice by a transient source of exogenous insulin

β-Cell plasticity governs the adjustment of β-cell mass and function to ensure normoglycemia. The study of how β-cell mass is controlled and the identification of alternative sources of β-cells are active fields of research. β-Cell plasticity has been implicated in numerous physiological and pathological conditions. We developed a mice model in which we induced major β-cell mass atrophy by implanting insulin pellets (IPI) for 7 or 10 days. The implants were then removed (IPR) to observe the timing and characteristics of β-cell regeneration in parallel to changes in glycemia. Following IPR, the endocrine mass was reduced by 60% at day 7 and by 75% at day 10, and transient hyperglycemia was observed, which resolved within 1 wk. Five days after IPR, enhanced β-cell proliferation and an increased frequency of small islets were observed in 7-day IPI mice. β-Cell mass was fully restored after an additional 2 days. For the 10-day IPI group, β-cell and endocrine mass were no longer significantly different from those of the control group at 2 wk post-IPR. Furthermore, real-time quantitative PCR analysis of endocrine structures isolated by laser capture microdissection indicated sequentially enhanced expression of the pancreatic transcription factors β(2)/NeuroD and Pdx-1 post-IPR. Thus, our data suggest this mouse model of β-cell plasticity not only relies on replication but also involves enhanced cell differentiation plasticity.

P62 Que devient la sous-unité Kir6.2 dans les cellules endocrines du pancréas de souris déficientes en SUR1 ?

Introduction Les sous-unites SUR1 et Kir6.2 contiennent dans leur sequence amino-acide un signal de retention qui controle leur association physique en un complexe hetero-octamerique formant les canaux KATP fonctionnels. Theoriquement, quand ces sous-unites ne sont pas co-exprimees ce signal de retention empeche leur migration vers la membrane cytoplasmique. Nous avons etudie le devenir de la sous-unite Kir6.2 en absence de SUR1 en comparant les cellules endocrines du pancreas de souris controle (WT) et de souris knockout pour le gene codant la sous-unite SUR1 (Sur1-/-). Materiels et methodes Les localisations sub-cellulaires de Kir6.2 et de la calreticuline (marqueur de reticulum) ont ete evaluees par des systemes de detections basees sur l’immunofluorescence en microscopie optique et sur l’immunogold en microscopie electronique. La longueur du reticulum rugueux lamellaire (L-RER) et la densite des particules d’or de Kir6.2 ont ete quantifiees par morphometrie. Resultats Comparee au WT, la surface de l’immunomarquage de la calreticuline est plus grande dans les cellules endocrines Sur1-/-, principalement autour des noyaux, ce qui reflete une plus grande densite du L-RER. L’immunolocalisation de Kir6.2 est superposable a celle de la calreticuline dans les 2 groupes et n’est pas observee a la membrane plasmique. En microscopie electronique, nous confirmons que le L-RER est plus abondant dans toutes les cellules endocrines chez les Sur1-/-, avec une longueur par surface de cellule endocrine 2 a 3x plus elevee, que chez les WT. La densite des particules d’or detectant Kir6.2 est aussi plus elevee dans le L-RER des cellules endocrines Sur1-/-refletant ainsi la sequestration de cette sous-unite dans cet organelle. Cependant, quelques particules d’or sont egalement observees dans les mitochondries et les granules de secretion. Conclusion En absence de SUR1, la majorite des sous-unites Kir6.2 est sequestree dans le L-RER mais une minorite echappe aux systemes de controle et se retrouve dans d’autres compartiments cellulaires en petite quantite.