The preference of fertilized (IVF) and somatic cell nuclear transfer (SCNT) presumptive zygotes for different media when cultured in vitro to the blastocyst stage was evaluated in this study. The experiment comprised two zygote production methods (IVF and SCNT) × two culture media (mSOF and G1.5/G2.5) factorial design in which culture droplets that contained approximate 30 presumptive zygotes formed the experimental plots for the assessment of cleavage and blastocyst development. There were 15 to 20 replicates (culture droplets) per treatment combination. Sub-samples 30 to 41 of the blastocysts produced were assessed for cell number and cell apoptosis. A further 10 blastocysts per treatment combination were used for quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the relative abundance of Hsp70 and Bax mRNA. Presumptive zygotes produced by IVF were developmentally more competent than SCNT zygotes in terms of cleavage rate (66.9 vs. 57.0%; P < 0.05) and blastocyst development rates (blastocysts of presumptive zygotes 29.7 vs. 24.8%; blastocysts of cleaved zygotes 44.4 vs. 36.6%; P < 0.05). Over both zygote production systems, however, the results were similar whether culture was in mSOF or in G1.5/G2.5 media for cleavage rate (63.2 vs. 62.4%; P > 0.05) and blastocyst development rate (blastocysts of presumptive zygotes 26.4 vs. 25.7%; P > 0.05; blastocysts of cleaved zygotes 41.8 vs. 41.2%; P > 0.05). There was, however, a significant interaction between the method of zygote production and culture medium for the apoptotic index of blastocysts. The interaction was such that IVF-produced zygotes cultured in mSOF had a lower apoptotic index compared with those cultured in G1.5/G2.5 (4.7 ± 1.2% vs. 9.8 ± 0.9%; P < 0.05) whereas SCNT zygotes had a higher apoptotic index when cultured in mSOF compared with those cultured in G1.5/G2.5 (11.9 ± 1.5% vs. 4.5 ± 1.2%; P < 0.05). Moreover, RT-PCR analysis showed that embryos from IVF-produced zygotes cultured in mSOF had a lower expression level of stress-related and apoptosis genes (Hsp70 and Bax) than those cells cultured in G1.5/G2.5 medium, while SCNT-derived embryos cultured in mSOF had a higher expression level of these genes than those embryos cultured in G1.5/G2.5 medium. The results of this study show that bovine IVF- and SCNT-produced presumptive zygotes have different nutrient requirements for in vitro culture to the blastocyst stage of development. IVF-derived zygotes have a preference for mSOF as the culture medium whereas the G1.5/G2.5 medium is more suitable for the culture of bovine SCNT-derived zygotes.

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

The present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities.

Although the sperm cryopreservation of freshwater and marine teleosts has been feasible for years, the cryopreservation of some fish embryos still remains elusive. Thus, the objective of this experiment was to analyze the embryo morphology after freezing and thawing 40 embryos of Piaractus mesopotamicus immersed into methanol and ethylene glycol, both at 7, 10 and 13% plus 0.1 M sucrose for 10 min. Soon after thawing, three embryos were treated with historesin, stained with hematoxylin–eosin and analyzed under an optical microscope. From every treatment, one palette containing embryos was thawed and incubated, but none of the eggs hatched. Samples containing two embryos were immersed into 10% methanol or 10% ethylene glycol both in association with sucrose, and embryos immersed into only water or sucrose solution were frozen, processed and analyzed using scanning electron microscopy (SEM). In both cases, the control group was immersed into only water. Although the embryos had the chorion, vitello, yolk syncytial layer and blastoderm, all of them were found altered under the optical microscope and by SEM. The chorion was irregular and injured; there was no individuality in the yolk granules; the yolk syncytial layer had an irregular shape, thickness and size; the blastoderm showed injuries in the nucleus shape and sometimes was absent; the blastoderm was located in atypical areas and absent in some embryos. In conclusion, no treatment was effective in preserving the embryos, and none of the embryos avoided injury from intracellular ice formation. These morphological injuries during the freezing process made the P. mesopotamicus embryos unfeasible for hatching.

Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

The objectives of this research were to study the influence of a reduced oxygen concentration during in vitro maturation (IVM) and examine the effect of follicular glucose concentration on bovine in vitro development and sex distribution. In the first experiment, abattoir-derived cumulus–oocyte complexes (COC) were matured under 5% O2 or 20% O2. Secondly, COC were isolated and the glucose (G) concentration of each follicle was determined. COC were pooled in groups (G<1.1mMol or G≥1.1mMol) according to the glucose content before being subjected to in vitro production (IVP). Cleavage and development rates were assessed on days 3, 7 and 8 post insemination. Blastocysts of each group were sexed by polymerase chain reaction (PCR). Expanded blastocysts were stained to assess total cell numbers and live–dead cell ratio. Cleavage and development rates stayed similar after reducing the O2 concentration during IVM. The sex ratio of embryos generated from oocytes matured under 5% O2 was shifted in favour of the female (♀: 61.9%), whereas the sex ratio of embryos belonging to the IVM 20% O2 group did not differ significantly from the expected 50:50 ratio. Neither a ‘higher’ nor a ‘lower’ intrafollicular glucose concentration influenced cleavage and development rates, cell numbers or live–dead cell ratio. Eighty five per cent (G<1.1) and 63.6% (G≥1.1) of the analysed embryos were female. In summary, neither a reduced O2 concentration during IVM nor selection based on follicular glucose concentrations affected the morphological quality of embryos. Although the sex distribution was shifted in favour of female embryos in all three experimental groups, more male embryos could be seen in the G≥1.1 group compared with the G<1.1 group.

The choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 106 and 7.4 ± 0.2 × 106 arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 106 arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.

Cryopreservation of germplasm provides a promising method to preserve fish genetic material, which is of great importance in preservation of species diversity, aquaculture, and management of fish models used in biomedical research. In the present study, cryopreservation of Rhinelepis aspera embryos, a Brazilian endangered species, was studied for the first time using a short-term cooling protocol. Embryos at blastoporous closing stage were selected, placed in 6-ml glass vials and stored at −8°C for 6 h in 10 different cryoprotectant solutions: S1 (17.1% sucrose + 9% methanol); S2 (17.1% sucrose + 9% DMSO); S3 (8.5% sucrose + 8.5% glucose + 9% methanol); S4 (8.5% sucrose + 8.5% glucose + 9% DMSO); S5 (17.1% sucrose + 9% ethylene glycol); S6 (8.5% sucrose + 8.5% glucose + 9% ethylene glycol); S7 (17.1% sucrose + 4.5% methanol + 4.5% DMSO); S8 (17.1% sucrose + 4.5% methanol + 4.5% ethylene glycol); S9 (17.1% sucrose + 4.5% DMSO + 4.5% ethylene glycol); and S10 (100% water). Embryo viability was assessed by hatching rate, counting live larvae and number of failed eggs under a stereomicroscope. The results showed that only the cryoprotectant solutions that contained methanol associated to sucrose (S1, S7 and S8) provided partial protection of Rhinelepis aspera embryos from cold damage (over 50% hatching rate in S1), while the use of DMSO and ethylene glycol, isolated or in combination, resulted in no hatching rate. Further studies are needed in order to extend the storage time and to improve the hatching rate for the species.

Oogenesis is a highly complex process that requires the exquisite temporal and spatial regulation of gene expression at multiple levels. Skin–embryo–brain–oocyte homeobox (Sebox) gene encodes a transcription factor that is highly expressed in germinal vesicle stage oocytes and that plays an essential role in early embryogenesis at the 2-cell stage in the mouse. As Sebox is also expressed in mouse fetal ovaries, the aim of the present study was to study its role during the early oogenesis in vitro. Expression of Sebox was low in 15.5 to 17.5 days post coitum (dpc) ovaries, showed a peak at 18.5 dpc and then its expression decreased dramatically in newborn ovaries. Sebox expression was efficiently knocked down (>80%) in fetal mouse ovary explants in culture using RNAi technology. When fetal ovary explants were transfected with Sebox-specific RNAi, the number of oocytes at germinal vesicle stage and showing a diameter of 40–70 μm was decreased significantly to 75% after 7 days of culture relative to the negative control, and to 22.4% after 10 days of culture, thus indicating that Sebox plays an important role in the early oogenesis in mice.

The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

One of the most important concerns in assisted reproduction (ART), and in particular ICSI, is the quality of sperm DNA. Oxidative stress is one of the major causes of damage to DNA and attempting to reduce generation of DNA damage related to reactive oxygen species (ROS) through consumption of antioxidants is often tempting. However, current antioxidant treatments, given irrespectively of clinically quantified deficiencies, are poorly efficient, potentially detrimental and over-exposure is risky. Here we discuss new treatments in relation to present day concepts on oxidative stress. This discussion includes stimulation of endogenous anti-ROS defense i.e. glutathione synthesis and recycling of homocysteine, the epicentre of multiple ROS-linked pathologies.

The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation.