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.

Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional KATP channels in all β-cells or only in β-cells within a resectable focal lesion. In more rare cases, without KATP channel mutations, hyperfunctional islets are confined within few lobules, whereas hypofunctional islets are present throughout the pancreas. They also can be cured by selective partial pancreatectomy; however, unlike those with a KATP focal lesion, they show clinical sensitivity to diazoxide. Here, we characterized in vitro IS by fragments of pathological and adjacent normal pancreas from six such cases. Responses of normal pancreas were unremarkable. In pathological region, IS was elevated at 1 mmol/L and was further increased by 15 mmol/L glucose. Diazoxide suppressed IS and tolbutamide antagonized the inhibition. The most conspicuous anomaly was a large stimulation of IS by 1 mmol/L glucose. In five of six cases, immunohistochemistry revealed undue presence of low-Km hexokinase-I in β-cells of hyperfunctional islets only. In one case, an activating mutation of glucokinase (I211F) was found in pathological islets only. Both abnormalities, attributed to somatic genetic events, may account for inappropriate IS at low glucose levels by a subset of β-cells. They represent a novel cause of focal congenital hyperinsulinism.

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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.

Peroxiredoxins are peroxidases involved in antioxidant defense and intracellular signaling. Expression of transcripts coding for peroxiredoxin 6 (PRDX6) has been previously described to be upregulated in oocytes after in vitro maturation, a period during which general transcription decreases dramatically in oocytes. The aim of the present work was to evaluate PRDX6 regulation in bovine cumulus-oocyte complexes in relation to maturation and intercellular communication. PRDX6 expression was analyzed by reverse transcription-PCR and Western blotting in oocytes and cumulus cells before and after in vitro maturation. PRDX6 was found to be upregulated at the mRNA and protein levels in both cell types after maturation. The effect of paracrine and gap junctional communication on PRDX6 expression was then assessed by culturing cumulus clusters in the presence or absence of denuded oocytes. While PRDX6 upregulation in oocytes required intact cumulus-oocyte junctions, the presence of denuded oocytes was necessary but sufficient for the upregulation to occur in cumulus cells. Finally, the influence of recombinant mouse growth differentiation factor-9 (GDF-9) on PRDX6 expression in cumulus cells was studied. GDF-9 induced cumulus expansion and PRDX6 upregulation in bovine cumulus clusters. Altogether, our data suggest that PRDX6 upregulation in cumulus-oocyte complexes during in vitro maturation is mutually regulated by both cell types: PRDX6 upregulation in oocytes would require gap junctions with cumulus cells, while upregulation in cumulus would depend on secretion of oocyte paracrine factor(s) with GDF-9 being a likely candidate.