Monitoring centriole dynamics in androgenetic sheep embryos

The centriole is a multifunctional organelle involved in chromosome segregation, cell cycle regulation, cell polarity, signalling and motility. Defects in centriole structure or function are associated with several human diseases, including cancer and male infertility. During early embryonic development centriole behaviour differs from that observed in somatic cells. Gametes must halve both their chromosome content and centriole number, and in most mammals, except for mice, the spermatozoon contributes the functional centriole to the zygote, whereas the oocyte centriole is inactivated. This ensures the presence of a single active centrosome during the earliest stages of embryogenesis. In dispermic fertilization, the entry of two spermatozoa introduces two centrioles, often leading to abnormal tripolar spindle formation and defective cleavage. Androgenetic embryos produced by fertilizing enucleated oocytes with two spermatozoa defeat this role, because, despite the presence of centrioles in double dose, they still divide and developed to the blastocyst stage, suggesting that one sperm-derived centriole may be repressed. These observations raise important questions regarding the mechanisms regulating centriole activation and inactivation during early embryonic development. The aim of this study was to investigate the behaviour of sperm-derived centrioles in ovine androgenetic embryos, focusing on the potential existence of functional “ON” and “OFF” states of centriole activity. Androgenetic embryos were produced by injecting two spermatozoa into enucleated metaphase II oocytes followed by in vitro activation. Embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were used as controls to establish the physiological time course of microtubule organization and aster formation after fertilization. Centriole activity was assessed by monitoring microtubule aster nucleation through immunofluorescence detection of α-tubulin, while chromatin and pronuclear formation were visualized with Hoechst staining and analysed by confocal microscopy. Analysis of androgenetic embryos approximately 6–7 hours after activation revealed heterogeneous patterns of microtubule aster nucleation. Two sperm asters were observed in 24% of embryos (n=5 on 21), indicating active microtubule nucleation from both sperm-derived centrioles. In 43% of embryos (n=9 on 21) only one detectable aster was present, suggesting asymmetric activation of the sperm centrioles, whereas in the remaining 33% (n=7 on 21) no asters were detected, indicating a possible repression of centriole activity at this stage. Interestingly, the proportion of one aster/androgenetic zygotes overlap with the cleavage rate (46%; n=18 on 39), suggesting that the inactivation of one centriole is a sine qua non condition for development. These preliminary findings suggest that sperm-derived centrioles in androgenetic embryos may exist in different functional states, supporting the hypothesis that regulatory mechanisms control centriole activation during the earliest phases of embryonic development.