Whole-depth change in bovine zona pellucida biomechanics after fertilization: how relevant in hindering polyspermy?
Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization 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.
Istituto di Fisica, Università Cattolica del Sacro Cuore, Roma, Italy.
Massimiliano
M
Papi
Roberto
R
Brunelli
Giuseppe
G
Familiari
Maria Cristina
MC
Frassanito
Luciano
L
Lamberti
Giuseppe
G
Maulucci
Maurizio
M
Monaci
Carmine
C
Pappalettere
Tiziana
T
Parasassi
Michela
M
Relucenti
Lakamy
L
Sylla
Fulvio
F
Ursini
Marco
M
De Spirito
D000818:Animals; D001696:Biomechanical Phenomena; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro; D005407:Flagella / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D008297:Male; D018625:Microscopy, Atomic Force / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D015233:Models, Statistical; D009865:Oocytes / Q000166:cytology / Q000648:ultrastructure; D012430:Ruthenium Red / Q000494:pharmacology; D013094:Spermatozoa / Q000166:cytology / Q000473:pathology*; D015044:Zona Pellucida / Q000378:metabolism* / Q000648:ultrastructure
D006023:Glycoproteins; D012430:Ruthenium Red