The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.     

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.     

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.     

Carbohydrates and fertilization in animals

A frequently used mechanism for sperm-egg recognition in many species involves complementary protein-carbohydrate interaction. The usual paradigm includes complex glycoconjugates in reproductive tract fluids or on the eggs which are recognized by carbohydrate-binding proteins on the sperm surface. Various glycoconjugates are utilized in the steps of sperm capacitation, sperm binding to the egg extracellular matrix and vitelline membrane and induction of the acrosome reaction. Several types of complex glycoconjugates are involved in these processes, including proteoglycans, lactosaminoglycans, sulfated fucose-containing glycoconjugates, and glycoproteins. There appear to be some structural similarities between active glycoconjugates; they are large in molecular weight and complex, and they are often sulfated, fucosylated, and attached to a protein through serine or threonine residues. In some species, the protein core of the glycoconjugates also participates in the interaction by limiting the binding of carbohydrates to sperm only of the relevant species, likely by providing the proper steric arrangement for the interaction. In other cases the protein core seems to serve more as a crosslinker of the carbohydrate moieties. This review discusses the types of glycoconjugates implicated in fertilization and the complementary lectin-like proteins found on sperm.     

Allelic polymorphism in the hamster oviductin gene is due to a variable number of mucin-like tandem repeats

Oviductins are high-molecular-weight glycoproteins specifically secreted by the oviduct. These proteins bind to the zona pellucida of the ovulated oocyte and remain associated with the embryo during its transit in the oviduct. They may be involved in fertilization and early embryonic development. In order to explore their putative biological function, the cDNA sequence corresponding to oviductin in the golden hamster was determined. We found that the deduced amino acid sequence of this heavily O-glycosylated protein presents characteristics typical of mucins, including serine- or threonine-rich tandem repeats. Analysis of several cDNA clones and of genomic DNA revealed the presence of a single copy gene with two frequent alleles differing in the number of repeats. Comparison with oviductin sequences from other mammals indicates a high degree of conservation amongst species, except for the repeat region which shows divergence, notably in the number of repeats. Based on its biochemical and genetic properties, hamster oviductin can now be classified as a secretory mucin. This concept provides a new insight in the elucidation of its biological role: oviductin could possibly provide the oviduct and the oocyte with a protective coating ensuring normal tubal function and embryonic development. © 1995 wiley-Liss, Inc.     

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.

Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).

The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.     

EVOLUTIONARY RELATIONSHIPS BETWEEN FOUR SPECIES OF CLADOPHORA (CLADOPHORALES,CHLOROPHYTA) BASED ON DNA–DNA HYBRIDIZATION1

Analysis of the reassociation kinetics of the DNA from Cladophora pellucida (Huds.) Kütz. indicates that the genome of this benthic alga is comprised of approximately 75% repetitive sequences. Single-copy sequences reassociated with a rate constant of 1.8 × 10^?3 M^?1· s^?1, which corresponds to a haploid genome size of 4.7 × 10⁸ bp. Genotypic relationships between members of the form section Longiarticulatae were determined by the method of DNA–DNA hybridization. No significant divergence was observed between the single-copy sequences of C. pellucida isolates from the East Atlantic coast and Mediterranean Sea. Cladophora feredayi Harv. and C. att. ad pellucida from Australia and C. pellucidoidea van den Hoek from the West Atlantic coast were highly and about equally divergent from C. pellucida. The data support the hypothesis that the West Atlantic–West Pacific divergence reflects the middle Miocene closure of the Mediterranean–Indo-Pacific seaways, and the hypothesis that the Northwest Atlantic–Northeast Atlantic divergence reflects the middle Miocene thermal separation of these coasts.     

哺乳动物透明带糖蛋白及其生物活性

透明带在哺乳动物的受精过程中发挥着重要作用,透明带化学成分及其生物活性的研究对于了解精卵识别、顶体反应的诱导等过程的分子机理具有重要意义．近10年来的研究表明透明带主要由糖蛋白（ZPGPs）组成,是在细胞质内合成后转移至透明带的.大多数动物的ZPGPs为4～5种,不同种类动物的ZPGPs的氨基酸序列存在高度同源性.在受精过程中,ZPGPs具有精子受体和诱导顶体反应的双重作用,ZPGPs含有N－连接和O－连接寡聚糖,这些寡糖链是ZPGPs行使生物活性不可缺少的部分．