Zona Pellucida Of Mouse Oocytes Research Articles

The Development of Piezo-Driven Tools for Cellular Piercing

Conventionally, intracytoplasmic sperm injection (ICSI) in the mouse is conducted with piezo-drills that use a droplet of mercury for damping. The use of mercury causes concerns of toxicity and contamination. Although Fluorinert can be used as a substitute for mercury to reduce piezo-drill’s lateral vibration, the damping effect is not as satisfactory as mercury. In this work, a modified piezo-drill without using mercury was developed for the cellular piercing of mouse oocytes. Experimentally, appropriate parameters of driving voltage and frequency were obtained for the penetration of the zona pellucida of mouse oocytes. Furthermore, the lateral vibration of the injection pipette is lower than 1 μm in deionized water, which is not observable at 400 magnificence. With the piezo-drill without using mercury, the system performs the cellular piercing of mouse oocytes with a maximum cleavage rate of 94.7% (n = 117).

Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone

Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of ‘promiscuous’ T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830–844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307–346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94–104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362–383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine.

The multifunctional zona pellucida and mammalian fertilization

An early step in mammalian fertilization is species-restricted binding of sperm to the oocyte's zona pellucida (ZP), a thick extracellular coat. Sperm bind to the ZP of unfertilized oocytes, but not to the ZP of fertilized oocytes. Shortly after binding to the unfertilized oocyte ZP, sperm undergo the acrosome reaction (AR). Three mouse ZP glycoproteins, called mZP1-3, constitute the mouse oocyte's ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized oocyte mZP3 at nanomolar concentrations are inhibited from binding to oocytes and undergo the AR; mZP1 and mZP2 have no effect. mZP3 from fertilized oocytes has no effect on sperm binding and is unable to induce the AR. These properties of mZP3 strongly suggest that it is a receptor for sperm and inducer of the AR. Mapping of the mZP3 combining-site for sperm suggests that it is located near the C-terminus of the polypeptide, in a region encoded by exon-7 of the mZP3 gene. This region of mZP3 is a site of positive Darwinian selection. When mZP3 exon-7 is fused to the Fc fragment of human IgG and sperm are exposed to the chimeric protein, the sperm are inhibited from binding to oocytes, suggesting that the inhibitory activity of mZP3 is dependent on this region of its polypeptide.

Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 m diode laser beam

The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 microm diode laser has been investigated by determining the ability of mouse oocytes to fertilize in vitro and develop in vivo. Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocyte-cumulus complexes. Pups derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. In conclusion, the 1.48 microm diode laser allows safe microdrilling of the zona pellucida of mouse oocytes after decoronization with hyaluronidase. Based on the health of the F2 generation and the lack of neuroanatomical and neurochemical differences, we concluded that this technology may be investigated in the human, particularly when the zona pellucida represents the main impediment for fertilization or embryo hatching.

Chromosomal constitution of mouse blastocysts derived from oocytes inseminated by multiple sperm insertion into the perivitelline space

Our purpose was to evaluate the rate of chromosomal aberrations in mouse blastocysts obtained after microinjection of multiple spermatozoa under the zona pellucida of mature oocytes. Without detecting the appearance of pronuclei, the microinjected mouse oocytes containing two polar bodies were cultivated to the blastocyst stage and then analyzed cytogenetically. A chromosome study was carried out in a total of 109 blastocysts derived after microinjection of motile spermatozoa into the perivitelline space. Fifty-five blastocysts (50.5%) exhibited normal diploid chromosome complements, 30 (27.5%) showed different forms of mosaicism, and 24 (22%) exhibited haploidy caused by parthenogenetic activation. Compared to in vivo and in vitro control groups there was a significant increase in the parthenogenesis and mosaic forms of embryos produced by micromanipulation (P < 0.001). A total of 360 well-spread metaphases of 103 blastocysts was analyzed to determine whether the micromanipulation procedure increased the chance of aneuploidy. Aneuploid numbers of chromosomes were absent in all the metaphases analyzed. Mosaicism and parthenogenesis appear to be increased significantly following microinjection of multiple spermatozoa under the zona pellucida of mouse oocytes, and there was no evidence of aneuploidy.

The efficacy and safety of zona pellucida drilling by a 193-nm excimer laser

To examine the efficiency of argon fluoride excimer laser drilling of the zona pellucida of mouse oocytes in improving in vitro fertilization (IVF) at low sperm concentrations and to assess its safety. Oocytes obtained from (Balb/c x C57BL6)CB6F1 female mice were drilled by laser and divided into two groups: group I (89 oocytes) were inseminated with 10(5) sperm cells/mL, and group II (94 oocytes) were inseminated with 10(6) sperm cells/mL. Both groups' fertilization rate and development in vitro was compared with control oocytes that underwent the same preparation steps but no drilling (94 and 88 oocytes for group I and group II, respectively). The fertilization rate and the development in vitro of the laser-drilled groups is compared with that of the control. In addition, in vivo development of embryos generated from laser-drilled oocytes after transfer to pseudopregnant recipients is assessed. For both sperm concentrations, laser drilling significantly enhanced fertilization over control (67% versus 31% at 10(5) sperm cells/mL and 90% versus 54% at 10(6) sperm cells/mL). The development into the blastocyst stage after 96 hours of incubation was similar for both the laser-drilled and control groups at any sperm cell concentration. However, complete hatching at this point was significantly enhanced by the drilling procedure. Normal litters were obtained from the transfer of embryos developed from zona-drilled oocytes into pseudopregnant recipients. Excimer laser drilling enhanced IVF at low sperm cell concentration. The procedure is safe and did not interfere with embryo development in vitro or in vivo.

An intact zona pellucida is not necessary for successful mouse embryo cryopreservation

To determine the developmental potential of mouse embryos that underwent cryopreservation after micromanipulation of the zona pellucida. Gaps were produced in the zona pellucida of mouse oocytes or two-cell embryos by zona drilling with acid Tyrode's solution. Zona-drilled oocytes were fertilized in vitro and cultured to the two-cell stage. Two-cell embryos were frozen, thawed, and cultured to the expanded blastocyst stage. There was no difference in the rate of embryo survival post-thaw (248/318, 77% versus 288/345, 83.4%), or in the rate of development to the expanded blastocyst stage (91/248, 36.7% versus 88/288, 30.6%), between embryos that were zona drilled as oocytes and unmanipulated controls. Similarly, there was no difference in the rate of cryosurvival (206/217, 94.9% versus 168/187, 89.8%) or development to the blastocyst stage (154/206, 74.7% versus 132/168, 78.6%) between embryos that were fertilized in vivo and zona drilled at the two-cell stage and embryos that were unmanipulated. These findings indicate that small gaps in the zona pellucida, such as those that result from micromanipulation, do not significantly alter the ability of embryos to withstand cryopreservation.

Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity.

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high levels of reporter gene activity, suggesting that the element is not only necessary but also sufficient for expression from zona pellucida promoters. Oligonucleotides containing the conserved element from either Zp-2 or Zp-3 form DNA-protein complexes of identical mobility in gel retardation assays using extracts of oocytes but not other tissues. These data are consistent with the hypothesis that common factors binding to conserved element IV are involved in coordinate expression of the oocyte-specific Zp-2 and Zp-3 zona pellucida genes.

Oocyte-Specific Factors Bind a Conserved Upstream Sequence Required for Mouse Zona Pellucida Promoter Activity

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high levels of reporter gene activity, suggesting that the element is not only necessary but also sufficient for expression from zona pellucida promoters. Oligonucleotides containing the conserved element from either Zp-2 or Zp-3 form DNA-protein complexes of identical mobility in gel retardation assays using extracts of oocytes but not other tissues. These data are consistent with the hypothesis that common factors binding to conserved element IV are involved in coordinate expression of the oocyte-specific Zp-2 and Zp-3 zona pellucida genes.

Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes

The effect of lectins with different saccharide specificity (ConA, LCA, DBA, WGA and PNA) on enzymatic digestion of the zona pellucida (ZP) of mouse oocytes was studied. All lectins tested, except PNA, induced ZP hardening with different degrees of effeciency. Moreover, extensive ZP digestion with mixed exoglycosidase prevented “spontaneous” ZP hardening. These observations suggest that changes of the carbohydrate moieties can be involved in the hardening of the zona pellucida of mouse oocytes.

Technique for cellular microsurgery using the 193‐nm excimer laser

A new cell surgery technique has been developed to produce well-defined alterations in cells and tissue without detectable heating and/or other structural damage in the surroundings. The technique involves the use of an argon fluoride excimer laser, in the deep ultraviolet (UV) region of the spectrum at 193 nm, which is guided through a glass pipette filled with a positive air pressure. To demonstrate the method, holes were drilled in the zona pellucida of mouse oocytes. The diameter of the drilled hole was determined by the pipette tip size, and its depth by an energy emitted per pulse and number of pulses. Scanning electron microscopy of the drilled mouse oocytes showed uniform, round, well-circumscribed holes with sharp edges. Oocytes that had their zona pellucida drilled with this new method fertilized in vitro and developed to the blastocyst stage in a rate similar to that of control group. These results demonstrate the nonperturbing nature of this cold laser microsurgical procedure. In addition to the extension of our results for clinical in vitro fertilization purposes, such as enhancement of fertilization and embryo biopsy, there are wide-ranging possible uses of our method in fundamental and applied investigations that require submicron accuracy in cellular alteration.

Fetuin Inhibits Zona Pellucida Hardening and Conversion of ZP2 to ZPZ during Spontaneous Mouse Oocyte Maturation in Vitro in the Absence of Serum1

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.

Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+-free medium.

Cumulus cell processes remaining in the zona pellucida of mouse oocytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl-phalloidin. If the isolation of the oocytes is performed in Ca2+-free medium, the presence of such processes allows the entry into the cell of low molecular weight molecules (such as 5-6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions. Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+-free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.

Fertilization of zona‐drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.

“Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro culture. II. The effect of follicular fluid and glycosaminoglycans

AbstractWe have previously reported that when isolated mouse oocytes are cultured in vitro their zonae pellucidae (ZP) become increasingly resistant to solubilization by chymotrypsin (“spontaneous” ZP hardening).In the present paper we report that follicular fluid contains factors that totally prevent such hardening. Furthermore, medium conditioned by granulosa cells partially prevents hardening.The protection against ZP hardening offered by follicular fluid may be ascribed, at least in part, to its content in sulfated glycosaminoglycans heparin and chondroitinsulfate B which, when added to the culture medium at physiological concentrations, show a marked antihardening effect.

“Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro culture

AbstractCulture in vitro causes a slow, progressive hardening of the zona pellucida (ZP) of fully grown dictyate oocytes isolated from the mouse ovary. Hardening cannot be prevented by inhibitors of peroxidase or by a tyrosine analogue. Culture in anaerobic conditions is very effective in preventing ZP hardening. If the oocyte is cultured surrounded by its own follicle cells or in contact with cumuli oophori obtained from superovulated females, hardening is much reduced. The results suggest that the “spontaneous” hardening in cultured ovarian oocytes is not due to a cortical reaction, and that a diffusible factor is produced by follicle cells that protect the ZP from hardening.

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

The zona pellucida is an acellular coat which surrounds the plasma membrane of fully grown mammalian oocytes and which performs a variety of important functions during oogenesis, fertilization, and preimplantation development. In this investigation the proteins of the mouse oocyte's zona pellucida have been identified and characterized by using zonae pellucidae isolated individually from fully grown oocytes with mouth-operated micropipets. Various morphological and biochemical criteria were employed to assess the purity of the isolated zonae pellucidae and, in most cases, they were found to be virtually free of contamination by other oocyte proteins. It was determined that each zona pellucida contains 4.8 ng of protein, which represents 80% or more of the dry weight of the zona pellucida and about 17% of the oocyte's total protein. Electrophoretic analyses of as few as five isolated zonae pellucidae treated with diazotized [ 125I]iodosulfanilic acid revealed the presence of only three radiolabeled proteins, designated ZP1, ZP2, and ZP3. The same three proteins were identified by Coomassie blue staining when large numbers of isolated zonae pellucidae (approximately 750) were subjected to SDS-polyacrylamide gel electrophoresis. These three proteins migrate as broad bands on SDS-polyacrylamide gels, consistent with their being glycoproteins, with apparent molecular weights of 200,000 (ZP1), 120,000 (ZP2), and 83,000 (ZP3). The same proteins were radiolabeled when intact oocytes were treated with diazotized [ 125I]iodosulfanilic acid, a reagent which does not penetrate the oocyte's plasma membrane, or when isolated zonae pellucidae were treated with 3H-labeled 1-dimethylaminonaphthalene-5-sulfonyl chloride. Results of amino acid analysis and high-resolution two-dimensional electrophoresis of the individual proteins suggest that each protein represents a unique polypeptide chain. The proteins ZP1, ZP2, and ZP3 represent about 36, 47, and 17%, respectively, of the total protein of the zona pellucida. In the presence of reducing agents which cause dissolution of the zona pellucida, ZP1 is converted into a species which migrates with an apparent molecular weight of 130,000, suggesting that it exists as an oligomer, stabilized by disulfide bonds, in the unreduced state. The results of these experiments are discussed in terms of the properties of the zona pellucida before and after fertilization and are compared with results obtained using vitelline envelopes of eggs from nonmammalian animal species.

走査電子顕微鏡による体外受精マウス卵子透明帯への精子侵入過程の観察

精巣上体尾部から採取し,約1時間前培養した精子と,hCG投与後16～17時間に採取した過排卵卵子を用いて,体外受精を行ない卵子透明帯への精子侵入過程を,走査電子顕微鏡を用いて経時的に観察した.授精後5分に固定した卵子(12個)のすべてに精子が見られ,またその半数の卵子で精子は透明帯への侵入を開始しており,11分では85%(17/20)の卵子で侵入途上にある精子が認められた.精子の透明帯通過によって生じる一端の切れこんだ,楕円形をした透明帯上の小孔(精子浸入孔)を有する卵子の割合は,11分での15%(3/20)から,15分の54(7/13),20分での80%(8/10)へと増加した.また,この精子侵入孔は授精後4時間を経たすべての卵子(19個)にも認められた.これらの結果から,精子は授精後5分までには卵丘を通過し,透明帯表面に達し,侵入を開始すること,また精子の透明帯通過は授精後20分以内にほとんどの卵子で完了することが知られた.さらに,侵入途上にある個々の精子の観察から,精子は酵素による透明帯物質の溶解と,頭部大彎部での機械的な切開によって透明帯を通過すると推察された.