The distribution of transglutaminase in the rat oocytes and embryos

Transglutaminases (TGs) are calcium-dependent enzymes that catalyze the transamidation of glutamine residues of a protein substrate to form intermolecular isopeptide bonds. The zona pellucida (ZP) is an extracellular, glycoprotein matrix that surrounds the oocytes of all Eutherian mammals. We aimed to identify the immunoreactivity of tissue transglutaminase (tTG) and ultrastructural changes occuring in rat oocytes before and after fertilization. Female rats were stimulated to superovulate, then mated with males. Oocytes and embryos were collected and examined by immunohistochemistry and electron microscopy. Before fertilization, tTG was present only in the oolemma and the cortical cytoplasm. After fertilization, tTG reactivity increased in the ZP of the early zygote and the preimplantation embryos, but decreased in the cytoplasm and perivitelline space (PVS). After fertilization, the PVS ultrastructure became asymmetrical and large around the polar bodies with many cortical granule contents. In conclusion, tTG immunoreactivity was found to be spatially and temporarily heterogeneous in the rat oocytes and embryos, especially in the ZP.     

Characterization, fate, and function of hamster cortical granule components

Little is known about the composition and function of mammalian cortical granules. In this study, lectins were used as tools to: (1) estimate the number and molecular weight of glycoconjugates in hamster cortical granules and show what sugars are associated with each glycoconjugate; (2) identify cortical granule components that remain associated with the oolemma, cortical granule envelope, and/or zona pellucida of fertilized oocytes and preimplantation embryos; and (3) examine the role of cortical granule glycoconjugates in preimplantation embryogenesis. Microscopic examination of unfertilized oocytes revealed that the lectins PNA, DBA, WGA, RCA(120), Con A, and LCA bound to hamster cortical granules. Moreover, LCA and Con A labeled the zona pellucida, cortical granule envelope, and plasma membrane of fertilized and artificially activated oocytes and two and eight cell embryos. Lectin blots of unfertilized oocytes had at least 12 glycoconjugates that were recognized by one or more lectins. Nine of these glycoconjugates are found in the cortical granule envelope and/or are associated with the zona pellucida and plasma membrane following fertilization. In vivo functional studies showed that the binding of Con A to one or more mannosylated cortical granule components inhibited blastomere cleavage in two-cell embryos. Our data show that hamster cortical granules contain approximately 12 glycoconjugates of which nine remain associated extracellularly with the fertilized oocyte after the cortical reaction and that one or more play a role in regulating cleavage divisions.     

Morphologic comparison of ovulated and in vitro–matured porcine oocytes,with particular reference to polyspermy after in vitro fertilization

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo–matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro–matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro–matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro–matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro–matured oocytes were dissolved within 131.7 ± 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro–matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for β-D-Gal(1–3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro–matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro–matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro–matured and in vivo–matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional block to polyspermy in pig oocytes. Mol. Reprod. Dev. 49:308–316, 1998. © 1998 Wiley-Liss, Inc.     

Biosynthesis of hamster zona pellucida is restricted to the oocyte

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian oocyte and the early embryo until implantation. This coat mediates several critical aspects of fertilization, including species-selective sperm recognition, the blocking of polyspermy and protection of the oocyte and the preimplantation embryo. Depending on the species, the ZP is composed of three to four different glycoproteins encoded by three or four genes. These genes have been cloned and sequenced for different species. However, controversy exists about the cell type specificity of the ZP glycoproteins, for which several models have been proposed. Different groups have reported that ZP is produced only by the oocytes, by the granulosa cells or by both cell types, depending on the species under study. We recently described the expression of four ZP proteins in the hamster ovary. By means of the complete set of the hamster ZP cDNAs, we undertook the study of the origin and expression pattern of the four ZP genes. In the present work, the expression of ZP1, ZP2, ZP3 and ZP4 is carefully analyzed by in situ hybridization (ISH) in hamster ovaries. Our data suggest that the four hamster ZP genes are expressed in a coordinate and oocyte-specific manner during folliculogenesis. Furthermore, this expression is maximal during the first stages of the oocyte development and declines in oocytes from later development stages, particularly within large antral follicles.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

p62/p56 are cortical granule proteins that contribute to formation of the cortical granule envelope and play a role in mammalian preimplantation development

The purpose of this study was to identify specific cortical granule protein(s) that form the cortical granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the cortical granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster cortical granules at fertilization and contribute to formation of the cortical granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the cortical granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as cortical granule proteins that contribute to the formation of the cortical granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.     

Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.     

Peptidylarginine deiminase (PAD) is a mouse cortical granule protein that plays a role in preimplantation embryonic development

Background  

While mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa cortical granule protein (p75) present in a subpopulation of mouse cortical granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.     

Mammalian fertilization: the egg's multifunctional zona pellucida

The zona pellucida (ZP) is a specialized extracellular coat that surrounds the plasma membrane of mammalian eggs. Its presence is essential for successful completion of oogenesis, fertilization and preimplantation development. The ZP is composed of only a few glycoproteins which are organized into long crosslinked fibrils that constitute the extracellular coat. A hallmark of ZP glycoproteins is the presence of a ZP domain, a region of polypeptide responsible for polymerization of the glycoproteins into a network of interconnected fibrils. The mouse egg ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3, that are synthesized and secreted exclusively by growing oocytes. One of the glycoproteins, ZP3, serves as both a binding partner for sperm and inducer of sperm exocytosis, the acrosome reaction. Female mice lacking ZP3 fail to assemble a ZP around growing oocytes and are completely infertile. Sperm bind to the carboxy-terminal region of ZP3 polypeptide encoded by ZP3 exon-7 and binding is sufficient to induce sperm to complete the acrosome reaction. Whether sperm recognize and bind to ZP3 polypeptide, oligosaccharide, or both remains an unresolved issue. Purified ZP3 self-assembles into long homomeric fibrils under non-denaturing conditions. Apparently, sperm added to ZP3 bind to the fibrils and are prevented from binding to ovulated eggs in vitro. These, as well as other aspects of ZP structure and function are addressed in this article.     

Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C57BL/6J X Mus spretus interspecific backcross progeny, Zp-2 was located on chromosome 7, 11.3 +/- 3.2 cM distal to Tyr, and Zp-3 was located on chromosome 5, 9.2 +/- 2.9 cM distal to Gus.     

Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.     

Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs and preimplantation embryos. Fertilization results in transformation of the zona pellucida (“zona reaction”), such that additional sperm are unable to bind to the zona pellucida of fertilized eggs and embryos, and sperm that had partially penetrated the zona pellucida of eggs prior to fertilization are prevented from further penetration after fertilization. The failure of sperm to bind to fertilized mouse eggs and embryos is attributable to modification of the sperm receptor, ZP3, an 83,000-molecular weight glycoprotein present in zonae pellucidae isolated from both eggs and embryos [Bleil, J. D., and Wassarman, P. M. (1980). Cell, 20, 873–882]. In this investigation, ZP2, the major glycoprotein found in mouse zonae pellucidae [Bleil, J. D., and Wassarman, P. M. (1980). Develop. Biol., 76, 185–202] was analyzed by gel electrophoresis under a variety of conditions in order to determine whether or not it undergoes modification as a result of fertilization. Under nonreducing conditions, ZP2 present in solubilized zonae pellucidae that were isolated individually from mouse oocytes, eggs, and embryos migrates on SDS-polyacrylamide gels with an apparent molecular weight of 120,000. However, under reducing conditions, ZP2 from embryos, but not from oocytes or unfertilized eggs, migrates with an apparent molecular weight of 90,000 and has been designated ZP2_f. The evidence presented suggests that modification of ZP2 following fertilization involves proteolysis of the glycoprotein, but that intramolecular disulfide bonds prevent the release of peptide fragments. It is shown that the same change in ZP2 can be generated in vitro by artificial activation of unfertilized mouse eggs with the calcium ionophore A23187, thus eliminating the possibility that a sperm component is responsible for the modification of ZP2 following fertilization. These results suggest that some of the changes in the biochemical and biological properties of zonae pellucidae, observed following fertilization or activation of mouse eggs, result from modification of the major zona pellucida glycoprotein, ZP2.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.     

Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa

The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6h after culture with or without (Control) oviductal cells (Experiment 1) and 3h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 +/- 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6h had a higher density than those pre-incubated for 3 h (P <0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes (P <0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.     

The ability of hamster oolemma to fuse with spermatozoa: its acquisition during oogenesis and loss after fertilization.

To determine when the growing hamster oocyte gains the ability to fuse with the spermatozoon, oocytes at various stages of development were collected from ovaries, and zona-pellucida-free oocytes were inseminated in vitro with acrosome-reacted spermatozoa. Very small primary oocytes were unable to fuse with spermatozoa. Oocytes first became competent to fuse with spermatozoa when they had grown to about 20 microns in diameter. The acquisition of fusibility coincided with the first appearance of zona pellucida material and oolemma microvilli. The fusibility of the oolemma increased as the oocyte grew, reaching a maximum when the oocyte reached the metaphase of the second meiosis. The fusibility of the oolemma was reduced drastically after fertilization, and was lost completely by the 8-cell stage. The appearance and subsequent disappearance of a putative fusion-mediating molecule in the oolemma is proposed. Since this molecule is fairly resistant to proteinase digestion, at least in the hamster, it could be a cryptic protein or a glycolipid.