Assembly of the Inner Perivitelline Layer, a Homo log of the Mammalian Zona Pellucida: An Immunohistochemical and Ultrastructural Study

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Cotumix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotennporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development. Copyright (C) 2011 S. Karger AG, Base

Egg extracellular coat proteins: From fish to mammals

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEa (~58 kDa), VEß (~54 kDa), and VEg (~47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (~200 kDa), ZP2 (~120 kDa), and ZP3 (~83 kDa). Overall, trout VE and mouse ZP proteins share ~25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEa, VEß, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEa and VEß are very similar in sequence (~65% sequence identity) and are related to ZP1 and ZP2, whereas VEg is related to ZP3 (~25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under growing oocytes in the ovary. The trout VE is assembled from VEa/g and VEß/g heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite ~400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the protein

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zonu pellirc.id(i (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZPI -3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the rnZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous ( r n ~ P 3 + / -o)r hornozygous ( r n ~ P 3 - / -f)o r the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female m ~ ~ 3 +m/ic-e are as fertile as wild-type animals, but their eggs have a thin ZP (-2.7 pm thick) as compared to the ZP (-6.2 pm thick) of eggs from wild-type animals. On the other hand, female rn~P3-/m- ice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated ~ZPJ-lfemales. Light micrographs reveal that development of ovarian follicles is often retarded in rnz~3- l -m ice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in I ~ Z P ~ -f/em- ales. I t seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells

Dynamic Oxygen Enhances Oocyte Maturation in Long-Term Follicle Culture

Traditionally, follicles have been grown in standard incubators with atmospheric oxygen concentration. However, preantral follicles exist in the avascular cortex of the ovary. This study examines the effectiveness of an oxygen delivery protocol that more closely mimics the in vivo environment of the ovary on oocyte viability, maturation, parthenogenetic activation, and fertilization from in vitro cultured rat preantral follicles. Of 54 oocytes cultured in the dynamic oxygen environment, 35 were viable while only 22 of 50 oocytes cultured within an ambient oxygen concentration remained viable (p < 0.05). Germinal vesicle breakdown was observed in 56% of oocytes from the dynamic oxygen group compared to 30% of oocytes from the ambient oxygen group (p < 0.05). Parthenogenetic activation was observed in a significant number of oocytes from the dynamic oxygen group, while none of the oocytes from the ambient oxygen group activated (p < 0.05). However, the proportions of oocytes from the dynamic oxygen group that remained viable underwent germinal vesicle breakdown, and activated were still significantly less than those from the in vivo control group (p < 0.05). Fertilization of the oocytes from the dynamic oxygen group was confirmed through a successful trial of intracytoplasmic sperm injection