New Insights into the Mechanisms of Fertilization: Comparison of the Fertilization Steps, Composition, and Structure of the Zona Pellucida Between Horses and Pigs1

Abstract

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin

“…Mugnier et al 2009). When performing such comparative studies, equatorin will be a good index of the acrosome reaction progression, because equatorin is conserved in all mammalian species examined so far including human (Yamatoya et al 2009;K Toshimori & C Ito, unpublished data).…”

“…zonadhesin (Hardy et al 1991, Gao & Garbers 1998, Olson et al 2004, ACRV1/SP-10 (Foster et al 1994), AM67/ZP3R/sp56 (Cheng et al 1994, Foster et al 1997, Kim et al 2001a, ZPBP1/sp38/IAM38 (Mori et al 1995, Yu et al 2006, AM50 (Westbrook-Case et al 1994, Kim et al 2001b, MC41 (Tanii et al 2002), SAMP32 (Hao et al 2002), ESP , SAMP14 , and acrosomal enzymes (reviewed by Moreno & Alvarado (2006)). In previous reports relating acrosomal molecules as described above, the status of the acrosome and the acrosome reaction progression were often measured by acrosome-related lectin stains such as Arachis hypogaea agglutinin (PNA), concanavalin A, and Pisum sativum agglutinin (Kallajoki et al 1986, Mortimer et al 1987, Holden et al 1990, Holden & Trounson 1991, Kinger & Rajalakshmi 1995, De Blas et al 2005, Harper et al 2008, Mugnier et al 2009, and in transgenic mice such as acrosin-green fluorescent protein (GFP) transgenic mice (Nakanishi et al 1999, Kim & Gerton 2003, Buffone et al 2009b. In human sperm, the acrosome reaction progression was identified as having six stages, with the intermediate ones based on transmission electron microscopic observations, in which acrosomal matrix material was lost while outer membranes appeared to retain their integrity (Stock & Fraser 1987, Yudin et al 1988.…”

Características do sêmen a fresco e descongelado de garanhões da raça Nordestina

“…As similaridades entre as glicoproteínas da zona pelúcida e da membrana perivitelina (MPV) do ovo de galinha, permitem a ligação dos espermatozoides de outras espécies a esta MPV (Mugnier et al 2009) e simula a interação dos gametas (Corcini 2010) sendo, portanto, um teste eficiente para se verificar a habilidade dos espermatozoides em sofrer a reação acrossomal (Corcini et al 2012). O uso da MPV do ovo de galinha, em relação aos ovócitos, é muito mais vantajoso porque permite, sobre diferentes fragmentos de uma mesma membrana, analisar diferentes partidas de sêmen, minimizando o efeito fêmea na análise e também por ser menos dispendioso que o ovócito (Csermak Junior 2011).…”

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

“…Equine sperm are able to bind firmly to ZP of bovine oocytes, undergo the acrosome reaction, penetrate the ZP, and fuse with the ooplasm (Sinowatz et al 2003). In contrast, Mugnier et al (2009) demonstrated that porcine ZP is more selective than equine ZP with fewer stallion sperm binding to porcine vs equine ZP. Based on these results, it appears that bovine ZP supports heterologous sperm binding and would be a better candidate for use in a heterologous ZP-binding assay.…”