168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

Abstract

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL^–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO_2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL^–1 fatty acid-free BSA, and 10 mg mL^–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 10⁶ spermatozoa mL^–1 at 39°C under 5% CO₂ during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO₂, 90% of N₂, and 5% O₂ atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.