Monitoring centriole dynamics in androgenetic sheep embryos: preliminary data​

In most mammals, the spermatozoon provides the only functional centriole, making its correct activation essential for successful cleavage during the first embryonic cell cycle. Although pronuclear formation occurs in approximately 80% of embryos produced via intracytoplasmic sperm injection (ICSI), cleavage rates remain significantly lower (~30%). This discrepancy, not observed under standard IVF conditions, suggests that post-fertilization developmental failure may be linked to centriole-dependent mechanisms. Among the proposed causes, defective sperm aster nucleation and abnormal microtubule organization have been identified as key factors impairing early embryonic progression. To investigate these mechanisms, we analyzed centriolar dynamics and sperm aster formation in ICSI-derived embryos using different chemical activation protocols, including ionomycin alone and ionomycin combined with cycloheximide. Sperm aster nucleation was assessed at multiple time points post-fertilization through α-tubulin immunodetection, revealing the critical role of the spermatozoon in organizing the microtubule network during syngamy in both IVF and ICSI embryos. We also employed sex-sorted ram spermatozoa (bearing either X or Y chromosomes) to assess whether sperm chromosomal sex influences aster formation efficiency, hypothesizing potential mechanistic differences between X- and Y-bearing sperm. In parallel, we produced an androgenetic embryo model—generated by injecting two spermatozoa into enucleated sheep oocytes—as a system to study centriole function in a completely paternal genomic context, enabling the isolation of sperm-specific regulatory factors. This study provides novel functional insights into sperm-derived centriole dynamics and represents an essential step toward identifying the molecular determinants of centriole activation, a key process required to initiate and support early embryonic development.