A simple and high-throughput method to assess maturation status of bovine oocytes: Comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4′,6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.     

Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.     

Fine structure of equine oocytes matured in vitro for 15 hours

Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microsocopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 ± 0.2°C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and these were scattered throughout the cytoplasm, whereas 15 hr oocytes contained numerous cortical granules primarily found in very close proximity to the oolemma. Further ultrastructural analysis of both groups revealed organelle structure similar to that previously described for in vivo matured equine oocytes. Evaluation of nucelar maturity (lacmoid stain) showed that 15 hr of culture resulted in significant numbers of oocytes at metaphase II (8/17; 47%). These data demonstrate that oocytes cultured for 15 hr in modified TCM 199 with 15% mare serum (day of ovulation) are mature with respect to nuclear configuration and cortical granule migration and, therefore, would be appropriate candidates for in vitro fertilization. © 1994 Wiley-Liss, Inc.     

Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure

The transmembrane protein bovine rhodopsin was reconstituted with egg phosphatidylcholine (PC) by using a modified detergent dilution technique employing the nonionic detergent octyl-beta-D-glucoside (octyl glucoside). Using this technique, reconstituted membranes having molar phospholipid/protein ratios between 60:1 and 255:1 were prepared. This is in contrast to the results obtained when an octyl glucoside dialysis technique was employed (Jackson, M.L. and Litman, B.J. (1982) Biochemistry 21, 5601-5608). In the latter case, the highest molar phospholipid/protein ratio that could be obtained when reconstituting rhodopsin with egg PC was approximately 50:1. Reconstituted vesicles prepared by the octyl glucoside dilution technique were examined by negative stain and freeze-fracture electron microscopy, and it was found that the vesicles were unilamellar providing the molar PC/protein ratio was below about 200:1, whereas in preparations having ratios higher than this, a significant number of the vesicles were multilamellar. The mean vesicle diameter showed no trend based on the molar PC/protein ratio within the range of 82:1 to 186:1. The mean diameters of the preparations were between 520 and 850 A. Approximately equal numbers of protein particles were observed on the concave and convex fracture faces of the freeze-fracture micrographs of the reconstituted membranes which is indicative of a symmetric distribution of the protein across the bilayer.     

Transillumination increases oocyte recovery from ovaries collected at slaughter. A new technique report

An inexpensive and convenient method of collecting large number of oocytes for in vitro procedures is by aspiration of follicles visible on the surface of isolated ovary. This method yielded only moderate numbers of oocytes per ovary, and it was found that the yield could be improved by slicing the tissue to reach deep, cortical follicles. However, slicing was time consuming and increased chances for sepsis. We developed a new technique that allows direct viewing of cortical follicles for aspiration of oocytes by transillumination of the ovarian medulla and cortex with a Plexiglas rod inserted through a small incision at the hilus. The technique, called "Transillumination-Aspiration Ovary" (TAO), increased the oocyte yield by 50% per ovary. The oocytes are probably recovered from deeper follicles which are difficult to identify during regular oocyte aspiration. The oocytes had a normal grading and exhibited normal in vitro development efficiency. Using the "TAO" technique we recovered 777 oocytes from 2160 follicles in 106 ovaries, a recovery rate of 36% from follicles and a mean of 7.3 oocytes/ovary. When we aspirated only surface follicles, we obtained 523 oocytes in 1384 visible follicles in 107 ovaries, for a recovery rates of 37% but a mean yield of 4.9 oocytes per ovary. Mean number of follicles were 20.5% with TAO and 12.8% without, thus recovery rates of oocytes per follicle were similar with both methods, but yield of oocytes per ovary was higher with TAO, thus showing that the difference between the two methods lies in higher numbers of visible follicles with TAO. Moreover, with the TAO technique 71% of the total oocytes we recovered (n=551) were grade I or II oocytes, in which 52% cleaved to the 2 to 4-cell stage and 26% had reached the blastocyst stage. We conclude that the method is effective for accurately locating cortical and peripheral follicles that contain oocytes suitable for IVF and in vitro embryo production (IVP).     

Banding patterns on lampbrush chromosomes of Triturus marmoratus (Amphibia Urodela) by the Giemsa stain

Lampbrush chromosome preparations from the newt species Triturus marmoratus have been submitted to a banding procedure by using a Giemsa stain technique (C-banding) as well as variants of the method. Centromeres, most of telomeres, the nucleolus organizing region and some segments along the chromosome axes appear to be differently stained. The centromere positions have been indicated on the maps of the lampbrush complement of the species. The possible relationships between banding and chromosome structure and organization are briefly discussed.     

Full term development of normal mice after transfer of IVF embryos derived from oocytes stored at room temperature for 1 day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 degrees C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 microg/ml 17beta-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p<0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.     

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.     

Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos

Cytogenetic analyses of mammalian eggs and preimplantation embryos have been limited by the difficult and tedious task of preparing chromosomes from single cells or small numbers of cells. In this report we describe a new technique that is both reliable and comparatively simple. Further, since the technique does not use the conventional 3:1 methanol:acetic acid fixative, it has the advantage of producing high-resolution chromosome preparations without destroying chromosome-associated proteins. Thus, this method provides a sensitive means of conducting studies of a heretofore inaccessible period of mammalian development, and of studying proteins thought to mediate both meiotic chromosome segregation and chromatin modifications in the preimplantation embryo.     

An Improved Method for Chromosome Preparations from Preimplantation Mammalian Embryos, Oocytes or Isolated Blastomeres

A method is described for making chromosome preparations from mammalian oocytes or preimplantation embryos, with or without the zona pellucida, or from isolated blastomeres. It is more robust and requires less skill and experience than previous techniques, yet chromosome structure is well preserved and very high quality preparations can be made. The method, which involves use of cold hypotonic solution and very cold fixative, reduces turbulence and allows even single blastomeres to be located and handled with relative ease, while the duration of hypotonic treatment becomes noncritical. The softening solution recommended contains no lactic acid and hence does not harm the chromosomes.     

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.     

Neuronal types in the lateral geniculate nucleus of man

Summary Nerve cell types of the lateral geniculate body of man were investigated with the use of a transparent Golgi technique that allows study of not only the cell processes but also the pigment deposits. Three types of neurons have been distinguished:Type-I neurons are medium-to large-sized multipolar nerve cells with radiating dendrites. Dendritic excrescences can often be encountered close to the main branching points. Type-I neurons comprise a variety of forms and have a wide range of dendritic features. Since all intermediate forms can be encountered as well, it appears inadequate to subdivide this neuronal type. One pole of the cell body contains numerous large vacuolated lipofuscin granules, which stain weakly with aldehyde fuchsin.Type-II and type-III neurons are small cells with few, sparsely branching and extended dendrites devoid of spines. In Golgi preparations they cannot be distinguished from each other. Pigment preparations reveal that the majority of these cells contains small and intensely stained lipofuscin granules within their cell bodies (type II), whereas a small number of them remains devoid of any pigment (type III). Intermediate forms do not occur.     

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.     

Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations

Cell viability in probiotic preparations is traditionally assessed by the plate count technique. Additionally, fluorescent staining combined with epifluorescence microscopy or flow cytometry has been developed for the viability assessment, but the currently available assays are either laborious or require highly sophisticated equipment. The aim of this study was to investigate the applicability of a microplate scale fluorochrome assay for predicting the cell state of freeze-dried Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis preparations. In addition to viability assessment with LIVE/DEAD BacLight Bacterial Viability Kit, DiBAC(4)3 stain was used for the kinetic measurement of changes in bifidobacterial cell membrane functions during exposure to low pH. The microplate scale fluorochrome assay results on the viability and cell numbers of probiotic preparations correlated well with the results obtained with the culture-based technique and (with few exceptions) with epifluorescence microscopy. The assay was applicable also for the viability assessment of stressed (acid-treated) cells provided that the cell density in treatments was adjusted to the optimal measurement level of the fluorometer. The microplate scale fluorochrome assay offers a rapid and robust tool for the viability assessment of probiotic preparations, and enables also kinetic measurements.     

Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

Chromosomal R-banding with a monoclonal antidouble-stranded DNA antibody

Summary A monoclonal anti-DNA antibody (HB2) specific for poly dG-poly dC nucleotides was used to stain metaphasic lymphocyte or amniotic cell human chromosomes. HB2 fixation was revealed using either a peroxidase-or a rhodaminelabeled anti-mouse immunoglobulin antiserum. The staining pattern of the chromosomes was dependent on the HB2 concentration: R-banding could be observed at high antibody dilution. Previous trypsinization of metaphasic preparations demonstrated a precise and reproducible typical R-banding independent of the HB2 concentration. This technique appears to be an interesting alternative to other R-banding procedures. The specificity of the antibody allows a better understanding of the biochemical mechanism of R-banding.