A Comparative Study of Zona Escape In Utero and In Vitro

Colorado State University-Pueblo, Pueblo CO

Investigators

Linked publications, trials & patents

Abstract

DESCRIPTION (provided by applicant): The means of zona escape in vivo are poorly understood, as are the factors contributing to in vitro "hatching" from the zona pellucida. The objectives of this proposal are to obtain an understanding of the contributing factors of in utero escape and in vitro "hatching" from the zona pellucida. In order to accomplish these tasks a comparative study of these two phenomena must be undertaken. These phenomena are viewed as different events and are, therefore, intentionally separated for the purposes of unraveling the mechanisms of zona escape in utero. Past studies by Gonzales and Bavister (1995), Gonzales et al., (1996) and Gonzales et al., (2001) together strongly suggest that "hatching" from the zona pellucida in vitro is an artifact of the culture conditions. Further, these studies suggest that "hatching" in vitro may be representative of implantation rather than zona escape. The demonstration of a tightly regulated in vivo protease activity occurring during a finite interval of the zona-escape-time-window provides a suspect in the investigation of the protease activity of in utero zona loss (Gonzales et al., 2001). Therefore, initially using the hamster (Mesocricetus auratus) model, the objectives of this study are to: 1) obtain a purified sample of the protease activities found in utero and in vitro and demonstrate that these activities possess lytic activity upon the zona pellucida, 2) determine the inhibitor and substrate specificity of these protease activities from hatching blastocysts conditioned medium and from uterine flushes, 3) develop a uterine epithelial cell culture system and stimulate this system to produce the putative protease activity responsible for zona escape in utero, 4) isolate the gene/s encoding the in utero and in vitro enzyme activities responsible for zona loss, and 5) demonstrate the existence of similar protease activity in the primate. The data collected from this investigation will shed light on zona loss both in utero and in vitro and may also provide some insight to the mechanism of the invasive characteristics of trophectoderm. Finally, one of the intentions of this study is to extend this investigation to the non-human primate using the rhesus macaques (Macaca mulatta) as a model.