Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in Go phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25–45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These ‘pronuclei’, similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Two closely related species in the camarodont sea urchin family Strongylocentrotidae, Strongylocentrotus pallidus and Strongylocentrotus droebachiensis, have circumarctic ranges with overlapping depth distributions and spawning seasons. Both species show genetic differences between Europe and the Pacific in the nuclear sperm bindin gene, and S. droebachiensis also differs in mitochondrial DNA. Gamete recognition systems of allopatric populations may or may not have drifted apart, but are unlikely to have diverged by reinforcing selection. Geographic divergence in gamete compatibility may illustrate mutational biases and variability and hence possible initial stages of reproductive isolation.

We carried out fertilisation experiments with both species' gametes from Norway and from the Eastern Pacific Ocean. Serially diluted sperm were added to egg suspensions in factorial designs, and fertilisation success was assessed by counting the proportion of cleaving embryos. To separate the specificity of the acrosome reaction from subsequent sperm–egg binding steps, a subsample of sperm was pre-activated by performing the last dilution step in diluted egg jelly from conspecific females. Fertilisation successes were compared by logistic regression ANCOVAs over five, 4-fold sperm dilutions for each cross. The graphs shown here illustrate fertilisation success for one sperm concentration only.

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 μM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 μM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 μM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 μM) and roscovitine (50 μM) induced pronucleus formation more efficiently than did 6-DMAP (100 μM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 μM) and retinyl (RI; 0.282 or 2.82 μg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin–nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

In this study, we examined a modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. The mature mouse oocytes were first vitrified and then warmed up in a modified cryoloop vitrification medium [15% ethylene glycol (EG) + 15% dimethyl sulphoxide (ME2SO) + 5.8 mg/ml Ficoll 400 (F) + 0.58 mol/l sucrose (S)]. These oocytes were later studied along with fresh oocytes, which served as the control group.

Based on the post-warm-up incubation time, the oocytes in the study group were divided into three subgroups: 0 h, 1 h and 2 h. We then examined the configurations of spindles and chromosomes, the fragmentation of DNA, and the oocyte's ability to be fertilized and developed into blastocysts. By evaluating the vitrified oocytes' morphology, we confirmed that 601 out of 612 (98.2%) oocytes survived this protocol. The percentage of oocytes with normal spindle and chromosome configurations in the study groups 0 h, 1 h and 2 h were all quite similar to each other and not statistically different from that of the control group. Similar results were also observed in the percentage of oocytes containing fragmented DNA. The fertilization rate and blastocyst formation rate of the thawed oocytes were not statistically different from that of the control group either. However, if not handled properly (too much remnant medium on oocytes in the process of freezing or too long a time of oocytes in the vitrification medium before freezing), the cryopreserved oocytes could show dramatic difference from the control group in terms of the morphologically survival rate, the configuration of the spindles and chromosomes, and the DNA fragmentation. In conclusion, when followed correctly, this modified cryoloop vitrification protocol had little effect on the survival rate and development potential of mature mouse oocytes.

The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

Staurosporine, an inhibitor of protein kinase activity, causes premature intercellular flattening of blastomeres but does not induce their premature polarisation. The flattening induced is calcium dependent, is reversed transiently at mitosis and requires the continuing presence of the drug. Staurosporine also blocks the decompacting effect of phorbol ester on 8-cell embryos.

In this study a specific inhibitor of cyclin-dependent kinases (cdks), butyrolactone I (BL I), was used for activation of pig and cattle metaphase II (MII) oocytes. BL I at a concentration of 100 μM was able to induce activation of both pig and cattle MII oocytes in a manner dependent on exposure time; however, precise timing of BL I exposure was required for the best activation results. The optimum activation rates were obtained when cattle MII oocytes were treated for 5 h with BL I and subsequently for 3-11 h in control medium, and pig MII oocytes for 8 h in BL I and then for 8-16 h in control medium; the percentage of activated oocytes after such treatment varied between 55% and 74% and between 53% and 81% for cattle and pig oocytes, respectively. Shorter exposures to BL I led to re-entry of the oocytes to the metaphase state in 35-50% of oocytes, the remaining oocytes forming a pronuclear stage; longer exposure to BL I led to increased numbers of oocytes being abnormal or degenerated. The behaviour of histone H1 kinase and mitogen activated protein (MAP) kinase, also measured during the experiment, reflected the morphological changes in the oocytes: both were inactivated after BL I treatment, though the inactivation of histone H1 kinase occurred 2 h ahead of that of MAP kinase. However, in the oocytes treated for a shorter time with BL I, with the reoccurrence of condensed chromatin in proportion of the oocytes cultured in control medium after BL I treatment, both kinases became reactivated. Taken together, these results suggest the possibility of using BL I for activation and cloning experiments in both species.

Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear–cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine–yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and ‘corrected’ the gene expression patterns of yak iSCNT embryos.

Morphogenesis and identification of embryonic differentiation in porcine embryos are crucial issues for developmental biology and laboratory animal science. The current paper presents a study on the asynchronous development of hatched porcine embryos from days 7 to 13 post-insemination. Examination of semi-thin sections of the hypoblast showed that it had characteristics similar to those of the mouse anterior visceral endoderm during embryonic disc formation. Also, a cavity appeared in the epiblast, which was similar to a mouse proamniotic cavity. With the gradual disappearance of Rauber's layer, the cavity opened and contacted the external environment directly, all of which formed the embryonic disc. To confirm the differentiation characteristics, we performed immunohistochemical analyses and showed that GATA6 was detected clearly in parietal endoderm cells during embryonic disc establishment. OCT4 was expressed in the inner cell mass (ICM) and trophoblast of hatched blastocysts and in the epiblast during formation of the embryonic disc. However, OCT4 showed comparatively decreased expression in the posterior embryonic disc, primitive streak and migrating cells. SOX2 was present in the ICM and epiblast. Therefore, both SOX2 and OCT4 can be used as markers of pluripotent cells in the porcine embryonic disc. At the start of gastrulation, staining revealed VIMENTIN in the posterior of the embryonic disc, primitive streak and in migrating cells that underlay the embryonic disc and was also expressed in epiblast cells located in the anterior primitive streak. Together with serial sections of embryos stained by whole mount immunohistochemistry, the mesoderm differentiation pattern was shown as an ingression movement that took place at the posterior of the embryonic disc and with bilateral migration along the embryonic disc borders.

Fresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin–eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

This study was designed to determine: (i) the in vitro effects of different freezing rates on post-thaw semen quality of Mediterranean brown trout (Salmo trutta macrostigma) from the Biferno river; and (ii) the in vivo fertilization and hatching percentage of freezing rate giving rise to the best post-thaw semen quality.

Pooled semen samples were diluted 1:3 (v:v) in a freezing extender composed of 300 mM glucose, 10% egg yolk and 10% dimethyl sulfoxide (DMSO). The extended semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen surface (1, 5 or 10 cm) for 10 min to give three different freezing rates. Semen samples were thawed at 30°C for 10 s. The variables assessed after thawing were sperm motility, duration of motility and viability.

Our results clearly indicate a significant effect of freezing rate on post-thaw semen quality. Semen frozen 5 cm above the liquid nitrogen surface showed the best quality after freezing/thawing. Based on these in vitro data, 2 groups of 200 eggs were fertilized with fresh semen or semen frozen 5 cm above the liquid nitrogen surface. Fertilization and hatching rates recorded for eggs fertilized with frozen semen were significantly lower (25.4% and 22.5%, respectively) than the ones obtained using fresh semen (87.8% and 75.5%, respectively). An effective freezing protocol will allow for the creation of a sperm cryobank to recover the original population of Mediterranean brown trout in the Biferno river.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

5-AZA-2′-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 μM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and β-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic–nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.

Pluripotency-associated transcription factors (PATFs) modulate gene expression during early mammalian embryogenesis. Despite a strong understanding of PATFs during mouse embryogenesis, limited progress has been made in ruminants. This work aimed to describe the temporal expression of eight PATFs during both sheep and cattle preimplantation development. Transcript availability of PATFs was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in eggs, cleavage-stage embryos, morulae, and blastocysts. Transcripts of five genes were detected in all developmental stages of both species (KLF5, OCT4, RONIN, ZFP281, and ZFX). Furthermore, CMYC was detected in all cattle samples but was found from cleavage-stage onwards in sheep. In contrast, NR0B1 was detected in all sheep samples but was not detected in cattle morulae. GLIS1 displayed the most significant variation in temporal expression between species, as this PATF was only detected in cattle eggs and sheep cleavage-stage embryos and blastocysts. In silico analysis suggested that cattle and sheep PATFs share similar size, isometric point and molecular weight. A phenetic analysis showed two patterns of PATF clustering between cattle and sheep, among several mammalian species. In conclusion, the temporal expression of pluripotency-associated transcription factors differs between sheep and cattle, suggesting species-specific regulation during preimplantation development.

The objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus–oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 μM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 μM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB+ group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB− group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 μM BCB+ group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB− oocytes was also higher in the 13 μM BCB group (n = 28/91; 30.78%) when compared with BCB− COCs exposed to 20 μM (n = 3/62; 4.84%) or 26 μM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 μM BCB group was similar between BCB+ or BCB− oocytes. The 20 μM BCB group had a lower rate of nuclear maturation of BCB− oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 μM and 20 μM, and an incubation period of 60 min.