Large scale isolation of bovine and pig zonae pellucidae: Chemical,immunological, and receptor properties

A procedure is described for isolating milligram quantities of bovine and porcine zonae pellucidae, uncontaminated by follicle cells or their processes. On SDS-polyacrylamide gel electrophoresis the isolated bovine zona material formed one major glycoprotein band with an estimated molecular weight of approximately 100,000 daltons and two minor lower molecular weight components. The isolated pig zonae formed only one glycoprotein band with a molecular weight of approximately 62,000 daltons. Rabbit antisera raised against the isolated zonae were zona-specific and formed only a single precipitin line against the heat-solubilized zonae on immunoelectrophoresis. An adjuvant was not required for high-antibody titers. High titers were also obtained by injection of the dog and rhesus monkey. Anti-zona antibody was detected by immunofluorescence, zona-coating, double-immunodiffusion, and the inhibition of spermbinding to eggs, including those of human origin. Antigenic and sperm receptor properties were stable at 100°C for five minutes, but some activity was lost after longer exposure. The serum antibody produced by rabbits immunized with pig zonae was predominantly IgG and cross-reacted with the zonae of a variety of other species, including primates. Pregnancy was inhibited in female rabbits immunized with pig zona preparations.     

Production of chimeric calves by aggregation of in vitro-fertilized bovine embryos without zonae pellucidae

Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.     

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.     

Immunization of female rabbits with heat-solubilized bovine zonae: Production of anti-zona antibody and inhibition of fertility

Bovine zonae were solubilized by heating to 80°C in buffered saline. On injection of female rabbits with this solution, zona-specific antisera were produced that could be assayed on bovine eggs in vitro by immunofluorescence, coating, and inhibition of sperm-binding. When the latter was complete, the rabbits were found to be infertile. Immunofluorescence assays showed that the rabbit anti-bovine zona serum reacted as strongly with rabbit and rhesus monkey zonae as with homologous zonae. Strong cross-reaction was also observed with marmoset and dog zonae, but with hamster zonae the reaction was relatively weak.     

Isolation of bovine zonae pellucidae from ovaries with collagenase: Antigenic and sperm receptor properties

Zonae pellucidae were collected from bovine ovaries by chopping, dispersing the chopped tissue with collagenase, sieving through nylon mesh screens, and pipetting. The zonae were free of corona cell processes when examined under the scanning electron microscope. Solutions of zonae obtained with collagenase exhibited the same antigenic and sperm receptor properties as those obtained without enzyme treatment.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000°C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over −180°C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction. Mol. Reprod. Dev. 51:53–58, 1998. © 1998 Wiley-Liss, Inc.     

Differential expression of ZPC in the bovine ovary,oocyte, and embryo

Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished. Mol. Reprod. Dev. 49:435–443, 1998. © 1998 Wiley-Liss, Inc.     

Timing of meiotic progression in bovine oocytes and its effect on early embryo development

This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest. Mol. Reprod. Dev. 47:456–467, 1997. © 1997 Wiley-Liss, Inc.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development

Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.