Fundamental cryobiology of mammalian oocytes and ovarian tissue

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy. Unfertilized mature (MII) stage oocytes are more difficult to cryopreserve than cleavage stage embryos of the same species. Many factors contribute to this including the oocyte's surface to volume ratio, single membrane, temperature-sensitive metaphase spindle and zona, and its susceptibility to parthenogenetic activation and chill-injury. A completely different set of problems applies to primordial follicles. Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. The problem lies in the difficulty in producing mature oocytes from these primordial follicles. Better and/or more convenient cryopreservation procedures for both oocytes and ovarian tissue are being developed. This paper describes some of the advances in this area and outlines the relative merits and limitations of several currently available egg and ovarian tissue cryopreservation procedures.     

Replication of somatic micronuclei in bovine enucleated oocytes

Background

Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.

Methods

Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (?)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (?)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (?), Parthenogenetic (?) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).

Results

All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.

Conclusions

We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.

     

Bile salts and calcium absorption

1. The study of the effect of bile salts on enhancing calcium absorption in the rachitic chick has been extended to bile salts not present in chick bile, e.g. glycine conjugates and bile alcohol sulphates. 2. Bile and bile salts cause an increase in calcium absorption from sparingly soluble calcium hydrogen phosphate when compared with a suspension of calcium hydrogen phosphate in saline. 3. If the bile ducts of normal rats are tied the absorption of calcium from calcium hydrogen phosphate decreases but can be restored by giving bile salts with the calcium salt. 4. Bile salts increase solubility in water of the sparingly soluble calcium salts, phytate and phosphate at pH values between 6 and 8. 5. Bile salts increase the solubility in lipid solvents of calcium in approximately the same proportion as they increase the absorption of calcium from the gut. 6. The physiological role of bile in calcium absorption and its mode of action are discussed.     

Mitochondrial DNA copy number in bovine oocytes and somatic cells

Restriction endonuclease analysis and direct nucleotide sequencing of bovine mitochondrial DNA have revealed a high apparent rate of sequence divergence between maternally related individuals. One possible mechanism that would account for the high rate involves nonuniform amplification and/or segregation of mitochondrial DNA during development of the oocyte. We report here experiments which quantitate the amount of mitochondrial DNA in the bovine oocyte as compared to bovine somatic cells. Total DNA was isolated from purified oocytes, separated by agarose gel electrophoresis, and immobilized on nitrocellulose filters. Hybridization with the complete mitochondrial DNA genome or cloned mitochondrial DNA restriction fragments revealed a 100-fold increase in oocyte mitochondrial DNA as compared to somatic cells. Developing oocytes contained about 4.5 pg or 2.6 × 10⁵ copies per cell, whereas primary bovine tissue culture cells contained 0.045 pg or 2.6 × 10³ copies per cell. These experiments demonstrate directly the amplification of mitochondrial DNA in mammalian oocytes and are consistent with models which could generate mitochondrial DNA polymorphisms by unequal amplification of mitochondrial genomes within an animal.     

Repetitive calcium transients in hamster oocytes

Golden hamster oocytes show repetitive Ca2+ transients at fertilization: a propagating Ca2+ rise from the sperm attachment site in the first 2-3 responses and synchronous Ca2+ rise in the entire egg in the succeeding responses. Cyclic Ca2+ rises are produced in unfertilized eggs by an injection of GTP gamma S or continuous injection of inositol 1,4,5-trisphosphate (InsP3). Both InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) are observed in hamster eggs, associated with a refractory period of 1-2 min after a Ca2+ release. In addition, external Ca2+ is a prerequisite for maintaining the repeated Ca2+ transients. The conditions that are expected to alter Ca2+ influx affect the frequency of Ca2+ transients with little effect on each response. The fertilizing sperm causes an increase in Ca2+ permeability of the egg plasma membrane and an increase in Ca2+ sensitivity of CICR. Feedback inhibition through protein kinase C is observed in G-protein-mediated Ca2+ transients but this inhibition seems to operate rather tonically. A model of Ca2+ oscillation is proposed: basically a second messenger-controlled oscillator model. InsP3 as the rigger of Ca2+ release is continuously supplied while an elevated basal [Ca2+]i level due to Ca2+ influx provides a favourable condition for IICR and CICR as well as for recharging the Ca2+ pools ready to release Ca2+ again.     

Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: Challenges for fertility preservation

Cryopreservation of ovarian tissue and in vitro follicle maturation are two emerging techniques for fertility preservation, especially in cancer patients. These treatment regimes are opening up more options and allow for more suitable choices to preserve fertility according to the patient's specific circumstances. If these technologies are to become widely accepted, they need to be safe, easy to perform and must obtain favorable results. The generation of healthy eggs with the normal genetic complement and the ability to develop into viable and healthy embryos requires tight regulation of oocyte development and maturation. Novel freezing techniques such as vitrification, along with whole ovary cryopreservation and three-dimensional follicle cultures, have shown favorable outcomes. The scope of this article is to take a comprehensively look at the challenges still faced in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.     

Intercommunication between mammalian oocytes and companion somatic cells.

Cellular interactions in the mammalian ovarian follicle between its germ-line and somatic cell components are crucial for its development and function. These interactions are mediated by both membrane gap junctions and paracrine factors. Somatic cell-to-oocyte communication is essential for oocyte growth and the regulation of meiotic maturation. In particular, granulosa cells provide nutrients and molecular signals that regulate oocyte development. Oocytes, on the other hand, promote the organization of the follicle, the proliferation of granulosa cells, and the differentiation and function of cumulus cells, a subset of granulosa cells. Determining the nature of the oocyte-to-granulosa cell signals remains a key challenge for future work.     

Immuno-localization of ferritin polypeptides in oocytes and somatic tissue of the freshwater snails Lymnaea stagnalis L. and Planorbarius corneus L.

Summary Antibodies were raised in rabbits against the 19000 Mr and 24000 Mr polypeptides of snail ferritin from Lymnaea stagnalis L. Anti-24000 Mr polypeptide antibodies were purified by an affinity-purification step and were made monospecific for their antigen by preabsorption with the 19000 Mr antigen. These purified antibodies were then used for in situ detection of their respective antigens by the indirect immunofluorescence method. The 19000 Mr polypeptide was found widely distributed in tissues of both pulmonate snails investigated (Lymnaea stagnalis L. and Planorbarius corneus L.) with the most intense antigen-directed fluorescence in certain connective tissue cells, secretory cells of the midgut gland and Sertoli cells and epithelia of the gonadal acini. In contrast, the 24000 Mr polypeptide could be detected only in yolk platelets of vitellogenic oocytes. The results indicate that yolk and somatic cell ferritins differ in immunoreactivity and structure and, accordingly may differ in function.This investigation was supported by the Deutsche Forschungsgemeinschaft. I greatly appreciate the advice given to me by Drs. U. Mays and V. Riedel, Münster.     

Capacitative calcium entry mechanism in porcine oocytes

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx.     

A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue.

Osmium-pyroantimonate solutions for the precipitation of cations are unsuitable for use with delicate mammalian oocytes. A variant of the pyroantimonate technique employing a mixture of pyroantimonate and glutaraldehyde has been found to give successful and repeatable results if a fixation time of 4 hr is used. Calcium-containing antimonate precipitates were localized principally in nuclei, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and cytoplasmic processes of both oocytes and follicle cells, and along the plasma membrane in small oocytes. Deposits were also concentrated around the periphery of lipid droplets in the follicle cells. The presence of calcium in the precipitates was confirmed by x-ray microprobe analysis.     

植物钙信号系统与体细胞胚发生

植物细胞离体培养中的体细胞胚发生受多种内外因素的调控,其中激素对细胞分化,发育和形态建成起着关键的作用。大量研究表明,在激素的作用过程中,Ca^2 信号系统可能是重要的介导者之一。Ca^2 和CaM在植物合子胚和体细胞胚发生中都具有重要作用,其作用机理可能是植物激素通过Ca^2 第二信号系统直接或间接地调控基因表达而实现的。     

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.     

'Trigger' events precede calcium puffs in Xenopus oocytes

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP₃Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca²⁺ puffs believed to arise from several to tens of clustered IP₃Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca²⁺ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca²⁺ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP₃ receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP₃Rs within the cluster by a regenerative process of Ca²⁺-induced Ca²⁺ release. Puff amplitudes (fluorescence ratio change) are on average ~6 times greater than that of the triggers, suggesting that at least six IP₃Rs may simultaneously be open during a puff. Trigger events have average durations of ~12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ~3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP₃ concentration, although the proportion of puffs showing resolved triggers was greatest (~80%) at low [IP₃]. Because Ca²⁺ puffs constitute the building blocks from which cellular IP₃-mediated Ca²⁺ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP₃Rs within clusters.     

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

The four dimensions of calcium signalling in Xenopus oocytes

This review focuses on the inositol phosphate/Ca2+ signalling pathway in Xenopus oocytes. The known characteristics of the individual elements of this cascade--from the membrane receptors to the intracellular Ca2+ stores--will be covered. Based on this knowledge, a simple model will then try to account for the behaviour of the newly recognized oscillations of free intracellular Ca2+ and propagated Ca2+ waves. Finally, some of the potential physiological functions of the inositol phosphate pathway will be summarized. Although there is no systematic attempt to contrast the findings in the oocyte to those in other cells, the readers of this journal will not fail to notice a high degree of similarity. Although this may seem unexciting at first, it suggests that the inositol phosphate signalling pathway may be strikingly conserved across species.