Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

We have investigated whether culture in glycine-containing medium affects subsequent glycine transport by the specific transport system, GLYT1, which is the sole glycine transporter in fertilized mouse ova. When fertilized ova were maintained for 6 h in culture with a physiological level of glycine (1 mM), subsequent transport of radiolabelled glycine was decreased by 40% compared with fertilized ova that had been maintained in glycine-free medium. Kinetic measurements showed that the apparent glycine affinity was decreased after culture with glycine (Km increased from 0.20 to 0.41 mM), but maximal transport rate was unchanged (similar Vmax of 20 and 23 fmol/fertilized ovum/min). These findings could have reflected activation of GLYT1 by prolonged substrate starvation, similar to some other amino acid transport systems. However, our findings were instead consistent with the alteration in glycine transport being due to trapping of glycine within the zona pellucida resulting in competitive transport inhibition even after ova were removed from glycine-containing media. First, even very brief exposures to glycine resulted in decreased subsequent glycine transport rates, with a maximal effect apparent within ~6 min. Second, extensive washing (at least six) reversed the effect. Third, the effect was absent when zona-free fertilized ova were used. Thus, it appears that components of the external environment of preimplantation embryos may continue to affect transport kinetics for a period even after embryos are removed from environments that contain them.

Cryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

Micromanipulation of oocytes is time consuming during ICSI experiments; however the ‘time frame’ to manipulate oocytes without a drop in efficiency is not very wide due to the use of not completely matured and/or aged MII oocytes. Therefore, the aim of this work was to study the effect of a short roscovitine pretreatment for 5 h and two different IVM periods (5R + 40IVM or 5R + 45IVM) and a prolonged IVM time from 45 h (45IVM) to 50 h (50IVM) on parthenogenetic and ICSI embryo development, in order to fit the time frame to manipulate pig oocytes to the whole labour day session. In the first experiment, oocytes, pretreated with roscovitine and IVM cultured for 5 h, showed a similar nuclear stage as non-cultured oocytes and a significantly higher percentage of GVI-GVII oocytes compared with non-roscovitine treated oocytes cultured for 5 h in IVM conditions. When COC were cultured under the 5R + 40IVM system, nuclear maturation and cleavage rates after electrical activation were significantly lower than when COC were cultured under the 45IVM, 50IVM and 5R + 45IVM culture systems (54.2% vs. 72.6–76.8% and 58.8% vs. 81.4–88.3%, respectively). However, this difference was not statistically significant for parthenogenote blastocyst rate. No differences were observed in MII and in parthenogenote and ICSI embryo development among 45IVM, 50IVM and 5R + 45IVM experimental groups. In conclusion, under our conditions and using parthenogenetic and ICSI embryos, we observed that it is feasible to prolong the pig oocyte manipulation ‘time frame’ by at least 5 h with no significant drop in blastocyst rate.

The cumulus–oocyte–complexes (COCs) recovery rates with respect to reproductive status per sei (Balaenoptera borealis) and Bryde's (B. edeni) whales were determined in Experiment 1. The number of COCs recovered ranged from 16.0 to 30.6 and from 6.7 to 26.8 per sei and Bryde's whales, respectively. The effects of COCs grades and protein supplementation in embryo culture medium on development of in vitro fertilized (IVF) embryos were evaluated in sei and Bryde's whales in Experiment 2. The COCs were classified into either Grade A (COCs with five or more layers of compact cumulus cells) or Grade B (COCs with less than five layers of compact or expanded cumulus cells) before being cultured for IVM. The cleavage (12.0 to 19.5%), 4-cell (8.0 to 12.0%) and 8-cell (4.0 to 8.0%) formation rates in sei whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either fetal whale serum (FWS)- or bovine serum albumin (BSA)-supplemented medium. The cleavage (4.0 to 14.8%), 4-cell (0.0 to 7.5%) and 8-cell (0.0 to 2.6%) formation rates in Bryde's whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either FWS- or BSA-supplemented medium. The grade B oocytes cultured in FWS-supplemented medium developed to morula stage (1.1%) in sei whales. In conclusion, the present study indicates that IVF in sei whales is possible to achieve cleaved embryos developing to morula stage. This is the first in vitro embryo production attempt in sei and Bryde's whales.

Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

The aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

To find more morphological characteristics useful for discussion on aralian or non-aralian Panax affinity, pollen morphological diversity was comparatively analysed in P. ginseng, Aralia elata and Oplopanax elatus collected during their pollination periods. In the anthers of both the buds and open flowers, the pollen average diameter varied between some species-specific maximum and minimal measurement. However, the larger pollen grains were typically found in the buds whereas the smaller pollen prevailed in the open flowers, testifying to the pollen size diminution during anther maturation. Based on this finding, the subsequent examination of pollen according to size decrease was put into operation as a method of pollen modification for the study. The structural mechanisms of pollen metamorphosis were identified as not being species specific but rather universal. These mechanisms are suggested to be the shrinkage of the pollen vegetative cytoplasm, the intine enlargement, the deepening of three colporate apertures provided by exine sunken into enlarged intine areas, the aperture accretion as well as the transformation of the exine from thick/sculptured into thin/less sculptured. During ‘size-reducing metamorphosis’, the pollen grains changed dramatically, going through a species-specific set of intermediate morphs to the final species-specific morphotype. In P. ginseng this morphotype is round (diameter is about 16 μm), in A. elata it is round with a single projection (diameter is about 15 μm) and in O. elatus it is ovoid with a single projection (average diameter is about 18 μm). In addition, every species is peculiar in having the unique vegetative cytoplasm inclusions and individual construction of the largest pollen exine. From a phylogenetic perspective, these findings presumably add support to the option of equal remoteness of P. ginseng from A. elata and O. elatus. The characteristics found seem to be suitable for examination of Panax affinity, by the subsequent study of more Araliaceae representatives.

While the freezing techniques of mammal embryos have been providing promising results, the cryopreservation of teleostean eggs and embryos have remained unsuccessful up to now. Therefore, this work aimed to develop a procedure of cryogenic preservation of embryos of Prochilodus lineatus and to observe, at both structural and ultrastructural levels, the morphological alterations that took place after the application of freezing/thawing techniques. The embryos at the morula stage could not tolerate exposure to the cryoprotectants ethylene glycol monomethyl ether, propylene glycol monomethyl ether, methanol, dimethyl sulphoxide and propylene glycol, presenting 100% of mortality. Embryos at the 4- to 6-somites stage tolerated exposure to propylene glycol and dimethyl sulphoxide, and the results revealed no significant differences (α = 0.05) regarding survival from both treatments. None of the freezing, thawing and hydration protocols was effective on preserving embryo viability. The ultrastructural analyses of frozen and thawed embryos showed that cells from ectoderm, somites, notochord and endoderm were structurally intact, with well preserved nuclei and mitochondria. The yolk globules were able to tolerate the freezing process, but the yolk syncytial layer was unorganized, displaying an electron-dense and compacted appearance, collapsed reticules, nuclei with modified chromatin and ruptures on the plasmatic membrane at the contact zone with endoderm. It might be concluded that the procedures tested for freezing were unable to avoid the formation of intracellular ice crystals, leading to drastic morphological modifications and making P. lineatus embryos unviable.

Phenotype integrity is viewed as an indicator of cumulus–oocyte complex (COC) viability. The objectives of this study were: (a) to observe the influence of cumulus investment expansion on the nuclear chromatin configuration of canine oocytes matured in vitro; (b) to examine the relationship between cumulus cell (CC) expansion and its morphology after in vitro maturation (IVM); (c) to ascertain the influence of in vivo serum progesterone (SP) concentrations of ovary donors on oocyte nuclear maturation, CC phenotypes and degrees of CC expansion of in vitro matured COCs. After 48 h of IVM in modified TCM 199, CCs from grade 1 and 2 COCs were stained with propidium iodide. Oocyte chromatin configuration was visualized by Hoechst 33342 stain. Results showed that oocyte IVM was not influenced by degree of CC expansion (D1, D2 and D3) in COCs. From the CC types (C1, C2 and C3), number of C1 types was higher at D1 expansion and differed from those observed at D2 and D3 expansions. Additionally, rates of apoptosis in D1 CCs were lower than those observed in D2 CCs (p < 0.05). Oocyte nuclear maturation was not influenced by in vivo SP concentrations of ovary donors. On the other hand, D3 expansion prevailed in COCs from bitches at SP > 2.5 ng/ml (p < 0.001). Moreover, in vitro CC apoptosis was associated both with low (0–1 ng/ml) and with high (>5 ng/ml) in vivo SP levels. These findings indicate that morphology of CCs from in vitro matured dog oocytes gives valuable information on viability of COCs and could possibly be used as a parameter in predicting the quality of oocytes destined for in vitro fertilization (IVF) and their outcomes.

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 μM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 μs) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5–81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2–32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.

During oocyte maturation, the cumulus–oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.

Oogenesis is a critical event in the formation of female gametes, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the chromatin architecture in newt oocytes. Using fluorescence microscopy, as well as transmission electron microscopy (TEM), immunohistochemical method and RE-ChIP assay, some peculiar aspects of chromatin and chromosome organization and evolution in crested newt oogenesis were investigated. We focussed our investigations on detection of certain epigenetic modifications (H4 hyperacetylation, H2A ubiquitinylation and cytosine methylation) at the rRNA gene (18S–5.8S–28S) promoter region. Our findings suggest that there is an involvement of some epigenetic modifications as well as of linker histone variants in chromatin architecture dynamics during crested newt oogenesis.

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.