This study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.

Karyotyping and cell number estimates in preimplantation embryos from heterogametic (XY*) and homogametic (XX) females of the field mouse Akodon azarae were studied to determine whether XX-XY-XY* differences exist in the rate of preimplantation development. At the morula stage, XY embryos from heterogametic mothers had twice the mean number of cells compared with XX embryos. However, this difference in cell numbers was not seen between XX and XY embryos from homogametic mothers. In this case, mean cell numbers were similar despite embryos being XX or XY. Furthermore, the mean cell number for XX and XY morulae from homogametic females was comparable to that for XX embryos from heterogametic females. It is concluded that XY* embryos (which will develop into heterogametic females) show an accelerated rate of preimplantation development.

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring–summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.

Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field.

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.

Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.

We have recently shown that the incubation of Xenopus laevis oocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16h, but it was not detected in those exposed for 24h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.

In vitro fertilized (IVF) human embryos have a high incidence of developmental arrest before the blastocyst stage, therefore characterization of the molecular mechanisms that regulate embryo development is urgently required. Post-transcriptional control by microRNAs (miRNAs) is one of the most investigated RNA control mechanisms, and is hypothesized to be involved actively in developmental arrest in preimplantation embryos. In this study, we extracted total RNA from mouse 2-cell and 4-cell embryos. Using a miRNA microarray, 192 miRNAs were found to be differentially expressed in 4-cell embryos and 2-cell embryos; 122 miRNAs were upregulated and 70 were downregulated in 4-cell embryos. The microarray results were confirmed by real-time quantitative RT-PCR for six miRNAs (mmu-miR-467h, mmu-miR-466d-3p, mmu-miR-292–5p, mmu-miR-154, mmu-miR-2145, and mmu-miR-706). Cdca4 and Tcf12 were identified as miR-154 target genes by target prediction analysis. This study provides a developmental map for a large number of miRNAs in 2-cell and 4-cell embryos. The function of these miRNAs and the mechanisms by which they modulate embryonic developmental arrest require further study. The results of this study have potential applications in the field of reproductive medicine.

In the following investigation the morphometric characteristics of the first two blastomeres of rainbow trout (Oncorhynchus mykiss) were determined. Embryos were incubated at 9°C and then fixed in a Stockard solution every 30 min starting from 8.5 to 12.5 h of incubation post fertilization. Embryonic discs were extracted and microphotographs were taken with Q Capture Pro 5.0 software using a stereomicroscope Olympus SZX7. The average size of the blastodiscs was 941.22 ± 160.42 μm. The first cleavage finished after approximately 12 h of incubation. The first two blastomeres were regularly symmetrical in their morphology. Blastomere 1 had an average length (L) of 942.68 ± 105.56 μm and width (W) of 467.34 ± 64.33 μm. Blastomere 2 had an average length of 887.60 ± 101.65 and width of 454.49 ± 47.25 μm (n = 91). Significant differences were found between the length and width of blastomeres 1 and 2. The proportion between the length of blastomeres 1 and 2 was 0.94 ± 0.07 (n = 91); between the width of blastomeres 1 and 2 it was 0.88 ± 0.11 (n = 91); and the width/length ratio was 0.51 ± 0.09 (n = 182). It was concluded that rainbow trout blastomeres tend to be asymmetrical in length with a higher dispersion of widths.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed ‘FLI’. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

The effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.

The study of nuclear molecular architecture during gametogenesis represents one approach towards the deciphering of the molecular organization of eukaryotic chromatin. During spermatogenesis, chromatin undergoes several dynamic transitions, which are often associated with important changes not only in its physical conformation but even in its composition and structure. Dynamic alterations in chromatin structure mediated by postsynthetic histone modification and DNA methylation constitute a major regulatory mechanism of gene function of eukaryotes. Using transmission electron microscopy and molecular investigations, some peculiar aspects of chromatin organization and evolution in spermatogenesis of the crested newt Triturus cristatus were investigated. We focused our investigations on the dynamics of chromatin structure after treatment with TSA (a histone deacetylase inhibitor).

The current study aimed to investigate differential expression of inhibin βA (INHβA) and inhibin βB (INHβB) in porcine oocytes before or after in vitro maturation (IVM) isolated from follicles of various sizes. Porcine oocytes isolated from large, medium and small follicles (40 from each) were used to study the INHβA and INHβB protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INHβA was found in oocytes collected from large and medium follicles compared with small follicles before or after IVM (P < 0.001, P < 0.05, respectively). Similarly, higher INHβB levels were observed in oocytes recovered from large follicles compared with small (P < 0.01). As INHβA and INHβB are expressed in both porcine follicular somatic cells and oocytes, it can be assumed that these transforming growth factor beta (TGFβ) superfamily factors are involved in the regulation of molecular bi-directional pathways during follicle and oocyte development, and can be recognized as markers of follicle and oocyte maturation. Moreover, the current study clearly demonstrated that inhibin expression is substantially associated with porcine follicle growth and development.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 μg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin–hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.

Following ovulation, oocytes undergo a time-dependent deterioration in quality referred to as post-ovulatory ageing. Although various factors influence the post-ovulatory ageing of oocytes, oxidative stress is a key factor involved in deterioration of oocyte quality. Artemisia asiatica Nakai ex Pamp. has been widely used in East Asia as a food ingredient and traditional medicine for the treatment of inflammation, cancer, and microbial infections. Recent studies have shown that A. asiatica exhibits antioxidative effects. In this study, we investigated whether A. asiatica has the potential to attenuate deterioration in oocyte quality during post-ovulatory ageing. Freshly ovulated mouse oocytes were cultured with 0, 50, 100 or 200 μg/ml ethanol extracts of A. asiatica Nakai ex Pamp. After culture for up to 24 h, various ageing-induced oocyte abnormalities, including morphological changes, reactive oxygen species (ROS) accumulation, apoptosis, chromosome and spindle defects, and mitochondrial aggregation were determined. Treatment of oocytes with A. asiatica extracts reduced ageing-induced morphological changes. Moreover, A. asiatica extracts decreased ROS generation and the onset of apoptosis by preventing elevation of the Bax/Bcl-2 expression ratio during post-ovulatory ageing. Furthermore, A. asiatica extracts attenuated the ageing-induced abnormalities including spindle defects, chromosome misalignment and mitochondrial aggregation. Our results demonstrate that A. asiatica can relieve deterioration in oocyte quality and delay the onset of apoptosis during post-ovulatory ageing.

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

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.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.