Mature Chorion Research Articles

Somatic Genomic Variations in Extra-Embryonic Tissues

In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.

Formation of mature egg envelope subunit proteins from their precursors (choriogenins) in the fish, Oryzias latipes: loss of partial C-terminal sequences of the choriogenins.

The inner layer of egg envelope of the medaka, Oryzias latipes, comprises two major groups of glycoprotein subunits, ZI-1,2 and ZI-3. Their precursor proteins, choriogenin H (Chg H) and choriogenin L (Chg L), respectively, are synthesized in spawning female liver. In the present study, the primary structures of the precursors and the corresponding mature subunits were compared by peptide mapping and amino acid sequencing to find what difference in their molecular structures is relevant to the assembly of the soluble precursors into the insoluble inner layer. The primary structures of the solubilized subunits were mostly identical to those of the respective precursors, but they lacked C-terminal partial sequences that their precursors possessed, namely, ZI-1,2 subunit was shorter than Chg H by 34 amino acid residues and ZI-3 was shorter than Chg L by 27 residues. In addition, a consensus amino acid sequence, Arg-Lys-X-Arg, was found at the putative cleavage sites in the C-terminal region of the precursors. It is conjectured that the truncation of the precursor proteins is prerequisite for formation of mature chorion subunit proteins and their assembly into chorion.

A Subtle Defect Underlies Altered Lamellar Orientation in the Gr16 Chorion Mutant of Bombyx mori

Gr 16 (chromosome 2, 6.9 cM) is a dominant, grey egg mutation of the domestic silkmoth, Bombyx mori, which produces an opaque instead of a translucent chorion. To determine the underlying cause of this defect, we examined the effects of the mutation on chorion ultrastructure, protein accumulation, and biosynthesis patterns, and on the restriction patterns of early chorion structural genes, using the presumed wild-type progenitor strain, Old European 16 ( OE16), for comparison. Cross-sections of mature chorions examined by scanning electron microscopy showed that the inner lamellae were oriented perpendicular, rather than parallel, to the egg surface, with a smaller vertical zone bordered by normally oriented lamellae in the heterozygote, suggestive of codominant expression. This defect first appeared in transmission electron micrographs early in choriogenesis, probably during framework formation and/or expansion stages. Mature chorion protein patterns resolved by isoelectric focusing or two-dimensional polyacrylamide gel electrophoresis, synthetic profiles of virtually all chorion proteins following pulse-labeling with tritiated or [ 14C]glycine, and restriction patterns of early chorion genes examined by Southern blot hybridization were identical between mutant, wild-type, and heterozygote. These observations ruled out a major deletion of chorion structural genes or defects in the production and accumulation of normal chorion proteins as mechanisms underlying the mutation, unlike other putative Gr mutants mapping in its vicinity. The sole difference that we detected was a set of minor, early protein doublets of related size but differing isoelectric point, which showed reciprocal labeling intensities in Gr 16 vs OE16 and were codominantly expressed. Preliminary pulse-chase experiments suggested that these proteins may be post-translationally modified forms which undergo differential processing in mutant and wild-type. Based on these observations, we postulate that the Gr 16 mutation affects the primary structure of a minor, previously unidentified chorion or follicular cell component which is critical for determining lamellar orientation, and discuss models of how this might function.

Fine structure of the chorion of Manduca sexta and Sesamía nonagrioides as revealed by scanning electron microscopy and freeze-fracturing

The fine structure of Manduca sexta and Sesamia nonagrioides chorion was investigated by scanning electron microscopy and freeze-fracturing. In both species the mature chorion exhibits a complex ultrastructure on its outer surface, with a large number of aeropyles forming polygonal arrays. The micropyle is surrounded by a rosette of approximately 80 follicular cell imprints. Scanning electron microscopy of vertically ripped sections reveals that both chorions consist of two main layers: a trabecular layer closest to the oocyte and a lamellar layer. The technique of freeze-fracturing, utilizing single-sided and rotary shadowing, clearly shows that fibrils, approximately 3–4 nm in diameter, constitute chorionic lamellae in both species. The fibrils appear to have a ‘beaded’ structure, with a 2–3 nm axial periodicity. Freeze-fracturing also provides a direct visualization of the helicoidal arrangement of these fibrils for the formation of chorion supramolecular architecture.

Ultrastructural analysis of chorion formation in the silkmoth Bombyx mori

The formation of the chorion in the silkmoth Bombyx mori (Lepidoptera) is analysed in detail by means of electron microscopy. The secretion of the proteinaceous chorion layers is being accomplished by the follicular epithelial cells which surround the oocyte. The mature chorion exhibits a tripartite ultrastructure; the trabecular layer closest to the oocyte, the inner lamellar layer, and the outer osmiophilic layer. The trabecular layer is deposited first upon the vitelline membrane and further modified possibly by rearrangement and (or) changes of its constituent proteins. A thin porous (sieve) layer is firmly attached to the tips of the follicle cell microvilli and appears to be implicated in the process of secretion of chorion proteins. In early choriogenesis, chorion is a thin (0.7–6 μm) lamellate and simply organized zone becoming thicker (12–20 μm) and more complex as choriogenesis proceeds. Chorion lamellae consist of helicoidally twisted fibrils embedded in an amorphous matrix. A denaturing agent (urea) was used to derive more information concerning assembly of chorion fibrous lamellae. The agent does not seem to eliminate the lamellar organization of chorion although it does modify it. There are suggestions that the agent disrupts many weak interactions, causing conformational changes in the structure of chorion protein components. The surface of the mature chorion is sculptured by follicle cell imprints. Small holes, the aeropyles, which extend radially into chorion down to the trabecular layer presumably serve for respiratory purposes. An elaborate depression in the anterior pole of the chorion, the micropyle, serves for sperm entry.

RNP particles at splice junction sequences on Drosophila chorion transcripts.

HnRNP particles are located at specific sites on nascent transcripts when chromatin is spread for electron microscopic visualization. To determine if the sequences bound by the particles play a role in RNA processing, we have correlated the nascent transcript morphology of Drosophila chorion s36-1 and s38-1 genes with their nucleotide sequences. We find that RNP particles about 25 nm in diameter are at the splice junctions of the introns in these two transcripts. On the more mature chorion transcripts, a single larger (40 nm) particle is occasionally seen in the same vicinity, which probably results from the coalescence of the two smaller particles. This RNP structure may be involved in bringing splice junctions into close proximity and in maintaining this proximity during the bipartite splicing intermediate stage.

X-ray diffraction studies of a silkmoth chorion

High- and low-angle diffraction studies have been performed on mature chorion (eggshell) of the silkmoth, Antheraea polyphemus. The results confirm the prevalence of β-sheet structure, previously suggested by predictions based on known primary structure and by results of laser Raman spectroscopy. The patterns obtained with different irradiation geometries suggest that a significant proportion of β-sheets are stacked and oriented with respect to the chorion surface and the ultrastructurally evident fibrillar components. Strong similarities are evident with the organization of β-sheets in chicken scale keratin.

Morphogenesis of silkmoth chorion: Initial framework formation and its relation to synthesis of specific proteins

Morphogenesis of the silkmoth eggshell is described at the ultrastructural level. Four zones are each assembled in a distinct manner and during a distinct developmental period: the innermost vitelline membrane and the adjacent trabecular layer appear consecutively, followed by a thin sieve layer, and a thick, lamellate chorion. Once formed, the sieve layer remains attached to microvilli, and thus all components which assemble into lamellae must pass through the sieve layer. Initially, lamellogenesis (and sieve layer formation) occurs in patches overlying trabeculae. Lamellae quickly fuse and new ones are added, presumably by apposition. Distinct types of lamellae seen in the mature chorion are already distinguishable in early lamellogenesis. The final lamellar number is attained before the developing chorion is one-half its final thickness or one-fifth its final dry weight. The early lamellae constitute a framework which is subsequently modified through expansion and densification. Proteins which may represent components of various parts of the eggshell have been identified on the basis of their timing of synthesis, relative amino acid compositions, and spatial distributions within the chorion.

Laser Raman studies of protein conformation in the silkmoth chorion

Laser Raman spectroscopy reveals extensive β-pleated sheet structure in a silkmoth chorion. This structure characterizes bath developing and mature chorion samples. α-helical structure is much less evident, but β-turns appear to be abundant. In the immature chorion the sulfhydryls are apparently found in diverse environments. The highly localized tyrosine residues appear to be strongly hydrogen-bonded in a hydrophobic environment.

Two clusters of genes for major chorion proteins of Drosophila melanogaster

cDNA clones complementary to poly(A)-containing RNA from ovarian follicle cells of Drosophila have been prepared. On the basis of restriction mapping and cross hybridization tests, 22 such clones were found to comprise four classes (group I–group IV). Filter-bound DNA from clones in each group hybridized, under stringent conditions, with a single size class of ovarian poly(A)-containing RNA. The mobilities of these RNAs on gels corresponded to those of putative chorion messenger RNAs (Spradling and Mahowald 1979). Translation of the RNAs in vitro yielded proteins with apparent molecular weights 1000–3000 daltons greater than those of mature chorion proteins isolated from purified eggshells. When a membrane fraction from dog pancreas was included in the in vitro translation reaction, the mobilities of the products were indistinguishable from those of major chorion proteins in three cases: c38 (group I), c36 (group II) and c18 (group III). The 16K product of hybrid-selected RNA from group IV clones could not be associated unambiguously with a specific chorion protein. Hybridization of cloned cDNAs to restriction digests of DNA prepared from embryonic nuclei was consistent with the presence of one (c38 and c36) or several (c18) copies of these genes per haploid genome. Hybridization in situ of RNA transcripts from the clones to polytene chromosomes demonstrated that the genes for these four proteins are clustered at two sites. In agreement with previous results (Spradling, Waring and Mahowald 1979), both c36 and c38 are coded at 7E11-7F1-2 on the X chromosome. Group III and group IV clones were both localized at a site on the third chromosome (66D15).

Formation of the vitelline membrane and chorion in developing oocytes of Ephestia kühniella.

Ultrastructural studies on developing egg chambers of the moth, Ephesita kuhniella reveal that the precursors of the vitelline membrane are synthesized within follicle cells which are in contact with the oocyte. The vitelline membrane precursors appear to be synthesized by the rough-surfaced endoplasmic reticulum, and this material apparently moves to the Golgi cisternae where the definitive vitelline membrane precursor body is produced. The previtelline bodies are then secreted into the spaces between the oocyte and follicle cells, where they fuse to form a continuous membrane. Chorion formation begins with the deposition of a layer of tubules at the outer edge of the vitelline membrane which coalesce to form the inner edge of a thin, striated layer. In subsequent stages, several compartmented layers are rapidly secreted external to the striated layer, giving rise to the mature chorion.

Fine structure of the chorion of a moth, hyalophora cecropia

The chorion of the moth Hyalophora cecropia has been found to consist primarily of a complex of proteins rendered insoluble by disulfide and hydrogen bonds. The bulk of the chorion is lamellate, and as in many cuticles each lamella contains a regular helicoidal series of microfibrils. The lamellar organization is disrupted in a central zone containing narrow irregular channels, and elsewhere the mature chorion is traversed by wide cylindrical channels. These open to the outer chorion surface indirectly via a porous trabeculate zone, and are distinct from the channels leading to the aeropyles, described elsewhere, in lacking cytoplasmic processes during their formation.