Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).     

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 beta-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 black to polyspermy in pig oocytes.     

Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.     

A review of major nursing vocabularies and the extent to which they have the characteristics required for implementation in computer-based systems

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread--as a result of cell fusion--to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2-3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte-zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the 'virgin' part of oolemma resistant to sperm penetration remains unknown.     

Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (Peroxisomal ghosts), representing a novel complementation group in mammals

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.     

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 zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice 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 mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice 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 mZP3-/- females. It 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.     

Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3(tm/tm)) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3(tm/tm) null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.     

p53 tumor-suppressor gene: clues to molecular carcinogenesis

Pig oocytes were examined to test their ability to undergo cortical granule exocytosis upon penetration by spermatozoa during meiotic maturation. Immature or maturing oocytes (cultured in vitro for 0 h, 26 h and 46 h) were inseminated with ejaculated boar spermatozoa in vitro. Before and after insemination, oocytes were stained with peanut agglutinin labelled with fluorescein isothiocyanate and the cortical granule distributions were examined under the fluorescent microscope and the laser confocal microscope. Before insemination, all the oocytes at the germinal vesicle stage showed a uniform distribution of cortical granules throughout the cortical cytoplasm. The granules migrated centrifugally during maturation and were distributed just beneath the oolemma in the oocytes after germinal vesicle breakdown, forming a monolayer in metaphase I or metaphase II. Cortical granules were still present in all penetrated oocytes at the germinal vesicle stage 18 h after insemination; in contrast, 26% and 84% of the oocytes inseminated at the stages of germinal vesicle breakdown or at metaphase I and II, respectively, completely released their cortical granules. Nuclear activation rates of penetrated oocytes were 0%, 38% and 96% in oocytes cultured for 0 h, 26 h and 46 h, respectively. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 88% (oocytes cultured for 46 h) released cortical granules completely. Complete cortical granule exocytosis was not observed in nuclear-inactivated oocytes. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 80% (oocytes cultured for 46 h) of monospermic oocytes and 67% (oocytes cultured for 26 h) and 91% (oocytes cultured for 46 h) of polyspermic oocytes released cortical granules, and no statistical difference was observed between oocytes cultured for 26 h or 46 h, or between monospermic and polyspermic oocytes. The proportion of oocytes with cortical granule exocytosis increased as insemination time increased and was greatest 18 h after insemination in oocytes cultured for 26 h and 46 h; no obvious changes were observed when the insemination time was prolonged to 24 h. These results indicate that pig oocytes develop the ability to release cortical granules after penetration by spermatozoa following germinal vesicle breakdown, and that this ability is not fully developed until metaphase II. Cortical granule exocytosis is accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic maturation are responsible for the cortical reaction. Polyspermy may be a result of a complete failure of cortical granule exocytosis in immature oocytes and delayed CG exocytosis in matured oocytes.     

The molecular genetics of the zona pellucida: mouse mutations and infertility

The zona pellucida is an extracellular matrix surrounding growing oocytes, ovulated eggs and the preimplantation embryo. After mediating the relatively species-specific events of fertilization, the zona pellucida provides a post-fertilization block to polyspermy and protects the growing embryo as it passes down the oviduct. The genes that encode the three zona pellucida proteins (ZP1, ZP2, ZP3) have been characterized in mouse and human. The ability to genetically manipulate the zona pellucida genes in mouse models has enhanced our knowledge of zona pellucida structure and function in vivo and may translate into a better understanding of human fertility.     

Timing of zona pellucida formation in the tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula)

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. The timetable of ZP formation was examined in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula) using conventional histological methods, immunofluorescence and electron microscopy. Ovaries from tammar wallaby pouch young less than 80 days of age contained only primordial follicles with a single layer of flattened granulosa cells. There was no evidence of ZP formation until 98 days, when a small number of eggs surrounded by a single layer of cuboidal granulosa cells had a ZP detectable by periodic-acid-schiff staining and rabbit anti-pig ZP polyclonal antibody labelling. Possum ovaries at 108 and 114 days also contained a small number of eggs with a ZP and a single layer of cuboidal granulosa cells. The antibody also labelled the peripheral cytoplasm of oocytes at this stage and, occasionally, the granulosa cells. Antral follicles were first detected at 144 days in the wallaby and 125 days in the possum, and always contained an egg surrounded by a ZP. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies contained a range of follicle types from primordial through early antrum formation. Electron microscopy confirmed observations made at the light microscope level. The ZP was first detectable in small primary follicles with a single layer of cuboidal granulosa cells in areas where microvilli had begun to form on the egg plasma membrane. Immunogold labelling indicated the egg cytoplasm as the origin of the ZP proteins. The ZP completely filled the space between the egg and the adjacent granulosa cells in preantral follicles, so that there was no perivitelline space.     

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.     

Activation of in vitro matured mouse oocytes arrested at first or second meiotic metaphase

Some mammalian oocytes fail to complete maturation in vitro and arrest development at the first metaphase stage. The response of such blocked oocytes to sperm penetration was investigated. Ovarian mouse oocytes from two inbred strains, CBA/Kw and KE, were cultured in vitro for 20 h. Both oocytes arrested at the first metaphase (MI oocytes) and second metaphase (MII oocytes) were then inseminated. The majority of MII and MI oocytes reinitiated meiosis in response to sperm penetration, although those from the CBA strain did with higher frequency. Moreover, a high proportion of unpenetrated oocytes from CBA, but not the KE strain, resumed meiosis (33% for MII and 48% for MI oocytes, respectively). Parthenogenetic activation of MI-arrested oocytes was demonstrated in (CBAxKE)F1 mice; ovarian oocytes matured in vitro and then treated by electric shock were activated with a similar total frequency of 52.4% for MI and 47.8% for MII oocytes. The rate of activation increased equivalently for both MI and MII oocytes as the length of maturation prolonged. This demonstrates that mouse oocytes arrested at MI during their maturation in vitro continue cytoplasmic maturation and become capable of undergoing activation in a way similar to those maturing to MII. Additionally, in MII oocytes cultured for an equal time in vitro the rate of activation increased with the time lapse after first polar body (PB1) extrusion. This indicates that after PB1 extrusion, the oocyte requires some resting time before it may be activated, perhaps to restore the proper balance between elements of the cell cycle controlling the mechanism involved in first meiotic division.     

Chemical modification and chemical cross-linking for protein/enzyme stabilization

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.     

rab3 mediates cortical granule exocytosis in the sea urchin egg

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 microns during maturation and increased to 106 microns after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination.     

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg

Exposure of mammalian oocytes to the protein phosphatase (PP)-1 (PP1) and PP2A inhibitor okadaic acid (OA) stimulates oocyte meiosis. However, treated oocytes do not develop beyond metaphase I (MI), and they display morphological aberrations. Experiments were conducted to define inhibitor treatment conditions for macaque oocytes that would result in germinal vesicle breakdown (GVB) stimulation and completion of meiosis without significant cytoplasmic abnormalities. As described above for OA, continual exposure of macaque oocytes to 50 nM calyculin-a (CL-A) significantly enhanced GVB at 24 h compared to that in controls, and the majority of the treated oocytes displayed cytoplasmic abnormalities. However, transient exposure (10 min) of rhesus macaque oocytes to either 50 nM CL-A or 1.0 microM OA enhanced GVB rates compared to that in controls and did not increase the incidence of cytoplasmic abnormalities. Meiotic maturation from germinal vesicle-intact oocytes to MII was enhanced following transient treatment with CL-A or OA compared to that in controls; however, development from MI to MII occurred at a similar frequency. In vitro-matured oocytes transiently exposed to OA and CL-A were capable of fertilization. In addition, ovarian immunohistochemical analysis revealed that both PP1 and PP2A were present in macaque oocytes. PP1 was localized throughout the cytoplasm with a predominance in the nucleus, whereas PP2A was evenly distributed throughout the cytoplasm with a reduction in the nuclear area. These results taken together-differential developmental responses to inhibitor treatment and intracellular enzyme localizations-may be indicative of multiple regulatory roles of PP1 and/or PP2A during meiosis.     

Preliminary evidence for aberrant cortical development in abused children: a quantitative EEG study

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 degrees C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8-15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents--CaA, ethanol, 6-DMAP, or cycloheximide--could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78-82% versus 3-13%) and blastocyst development (31-40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide-treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro-fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes.     

Structural and functional differentiation of follicular and oviductal mouse oocytes visualised with FITC-protein conjugates

The fluorescence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida, perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage (GV) ovarian oocytes exhibited a bright, amorphous, mesh-like staining pattern (immature type). In contrast, mature polar body stage (PB) oocytes, either ovarian or oviductal, displayed faint, spotty fluorescence labelling of the ZP (mature type). The perivitelline space (PVS) of mature ovarian oocytes (12 h post-hCG) failed to label, whereas approximately 50% of oviductal oocytes showed PVS labelling. The incidence of PVS staining increased with postovulatory age, possibly as a result of the accumulation of materials secreted by the oviduct. Following in vivo or in vitro fertilisation of oocytes, a characteristic pattern of plasma membrane (PM) labelling was observed. Similar patterns of PM labelling were seen in oocytes parthenogenetically activated with ethanol or ionophore (A23187) but not in control oocytes. The pattern of PM labelling observed with FITC-protein conjugates was strikingly similar to that observed with FITC-labelled lectins, which are thought to interact with glycoconjugates released from cortical granules. Immature type of ZP staining also occurred when GV oocytes were treated with FITC alone or with a variety of FITC-protein conjugates. Thus, protein may not be required for labelling of the ZP by FITC-protein conjugates as previously thought. FITC-conjugated proteins including casein, bovine serum albumin, peroxidase and non-immune immunoglobulin G (IgG), all labelled the PM of activated oocytes; however, FITC-IgG failed to label the PVS. Results demonstrate for the first time that various components of viable mouse oocytes exhibit and undergo characteristic structural and functional changes during periovulatory differentiation as evidenced by their interaction with one or more FITC-protein conjugates and/or FITC. On the basis of these results the intrafollicular and oviductal mechanisms mediating these changes are discussed as is the possibility that the fluorescent molecule attached to conjugates may play a role in oocyte labelling.     

Postnatal development of Leydig cells in the opossum (Monodelphis domestica): an immunocytochemical and endocrinological study

Using a mouse early preantral follicle culture system, mature full grown oocytes, arrested in prophase I of meiosis, were produced after 12 days using a recombinant gonadotrophin-supplemented medium. This culture medium does not mimic the normal extracellular environment of the oocyte and might therefore modify meiotic regulation and more particularly progression to metaphase II (MII). The aim of this study was to optimize the treatment using recombinant stimulatory ligands which were known to induce germinal vesicle breakdown (GVBD) and completion of meiosis I, metaphase II (MII), namely recombinant follicle stimulating hormone (r-FSH), chorionic gonadotrophin (r-HCG) and epidermal growth factor (EGF). Full-grown intrafollicular oocytes could not resume meiosis when the 'ovulatory' stimulus was r-FSH, used at a 100 times higher dose than during culture. r-FSH did not increase progesterone production. When 1.5 IU/ml r-HCG was used as meiotic trigger, germinal vesicle breakdown was obtained in 95% of the oocytes 64% of which extruded a first polar body. r-HCG induced a dramatic increase in progesterone production. When EGF was administered as sole stimulus on day 12 to the attached follicle-enclosed oocytes, only doses > or =5 ng/ml could cause GVBD, although less effectively than r-HCG (45 versus 95%; P < 0.0001). Oocytes undergoing GVBD by the EGF pulse reached metaphase II at a rate of 54% (not significant versus r-HCG). EGF did not stimulate progesterone production. Addition of increasing doses of EGF (0.5; 5; 10; 50 ng/ml) to r-HCG did not increase the GVBD-rate, but EGF doses >5 ng/ml improved MI to MII transition (P=0.027), thereby improving the final yield of MII oocytes by 12.5%. These data show that up to a dose of 50 ng/ml, EGF on its own could only override the somatic inhibitory stimuli in less than half of the cultured follicles. However, in addition to HCG, EGF (25 ng/ml) had a stimulatory effect on completing the first meiotic division. It was concluded that, under the present culture conditions, EGF in combination with HCG provided optimal nuclear maturation.