The dynamic structure of rabbit blastocyst coverings

Abstract

Under physiological conditions the zona pellucida disappears in the rabbit between Day 3 and early Day 4 post coitum (p.c.) and is replaced by a new layer, the neozona. The dissolution of the zona pellucida and the formation of the neozona was investigated in three different experimental approaches, all of them characterized by non-physiological developmental conditions for the embryo: Prevention of embryo migration from the oviduct into the uterus by postcoital (48 h p.c.) tubal ligation, in vitro culture, and asynchronous embryo transfer into uteri of recipient rabbits. Embryos of age 2 1/2, 3, 4 and 4 1/2 days p.c. were cultured for 12 to 72 h. The media used for in vitro culture were supplemented with BSA, serum or with uterine secretions that were collected either synchronously or asynchronously to the developmental stage of the cultured embryos. Three-day-old embryos were transferred into uteri of pseudopregnant foster rabbits of either synchronous (Day 3) or asynchronous stages (Day 0, 2, 4, 5, 6) and were recovered 24 to 72 h after transfer. The transformation of the coverings was evaluated by light and transmission electron microscopy. The dissolution of the zona pellucida was greatly disturbed in tube-locked embryos, and in cultured embryos if standard protein supplements (BSA or serum) had been used for in vitro culture. In many cases the zona was still completely preserved after 2 or 3 days in culture, at a time when it normally would have already been replaced by the neozona in vivo. The dissolution in vitro, however, progressed incomparably better if the culture medium had been substituted with synchronous or asynchronous uterine secretions. The formation of the neozona could not be verified in cultured blastocysts. After embryo transfer, the dissolution of the zona pellucida was completed in most cases by 2 days after transfer, irrespective of the recipients' progestational stage. Present results indicate that uterine components are essential for the dissolution of the rabbit zona pellucida. These components appear to be present in the uterine cavity constitutively, i.e. independently of the uterine progestational transformation, and need not be in synchrony with the embryo's developmental stage for dissolution of the zona. Normal formation of the neozona does not take place under the non-physiological developmental conditions of in vitro culture.

The Aggregation of Four Reconstructed Zygotes is the Limit to Improve the Developmental Competence of Cloned Equine Embryos

“…Thus, if present, it must be deficient. Alterations in early embryonic coats have been reported for equine ICSI [56] and cloned embryos [8] and also for rabbit [57] . Hence, improvements in in vitro culture conditions are necessary to allow for the normal formation of early embryonic coats in this species.…”

Rabbit as a reproductive model for human health

“…The rabbit embryo enzymatically dissolves the zona pellucida during day 4 (Denker & Gerdes 1979) and replaces it by the neozona during day 5 p.c. The peri-implantation blastocyst is covered by three extracellular coverings: the neozona, remnants of the mucoprotein layer deposited during tubal passage and uterine secretions (Denker 1977, Fischer et al 1991.…”

Influence of the uterine environment on the development of in vitro-produced equine embryos

“…In this respect, the equine embryonic capsule has been proposed to be most analogous to the neozona of the rabbit (Betteridge 1989, Herrler & Beier 2000. For example, while trophectodermal secretions are important for neozona formation, maternal components also appear to be critical (Fischer et al 1991, Denker 2000. Indeed, in vitro culture of rabbit embryos is not only associated with the deposition of granular material on the inside of the mucoprotein layer, but also with failure of normal neozona formation, aberrant herniation of embryonic cells through the zona, and incomplete or failed dissolution of the zona pellucida (Fischer et al 1991).…”

“…For example, while trophectodermal secretions are important for neozona formation, maternal components also appear to be critical (Fischer et al 1991, Denker 2000. Indeed, in vitro culture of rabbit embryos is not only associated with the deposition of granular material on the inside of the mucoprotein layer, but also with failure of normal neozona formation, aberrant herniation of embryonic cells through the zona, and incomplete or failed dissolution of the zona pellucida (Fischer et al 1991). These features are reminiscent of the aberrations of capsule formation and hatching observed in IVP equine embryos (Tremoleda et al 2003, Stout et al 2005.…”

“…of uterine flushings to rabbit embryo culture medium, or intrauterine transfer of in vitro-cultured embryos, has been shown to allow reactivation and completion of zona dissolution, although uterine flushings alone did not induce neozona formation in vitro (Fischer et al 1991). In the horse, intrauterine transfer of IVP blastocysts results in successful pregnancies (Galli et al 2007, Hinrichs et al 2007, Smits et al 2010 with an apparently normal capsule (Choi et al 2009), illustrating that the degree of initial disturbance to capsule formation during IVP is not so severe as to preclude normal pregnancy, at least as long as exposure to the uterine environment is sufficiently early to remedy the aberrations; by contrast, total removal of the blastocyst capsule from day 6.5 embryos is incompatible with embryonic survival after transfer (Stout et al 2005); clearly, the disruption to capsule formation suffered during IVP is not equivalent to removal.…”