Whole-Depth Change in Bovine Zona Pellucida Biomechanics after Fertilization: How Relevant in Hindering Polyspermy?

Massimiliano Papi, Giuseppe Maulucci, Marco De Spirito, Maria Cristina Frassanito, Roberto Brunelli, Giuseppe Familiari, Luciano Lamberti, Maurizio Monaci, Carmine Pappaletter, Tiziana Parasassi, Michela Relucenti, Sylla Lakamy, Fulvio Ursini

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)


Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this species, after IVF, despite the lack of a biochemical postfertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.
Original languageEnglish
Pages (from-to)e45696-1-e45696-7
Number of pages7
JournalPLoS One
Publication statusPublished - 2012


  • Atomic Force Microscopy
  • Zona Pellucida

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