The adenosine monophosphate-activated protein kinase (AMPK) activators, 5′-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.5, 1, 5, and 10 μM) did not reverse the inhibitory effects of AICAR (1 mM) and MET (2 mM) on bovine oocyte meiosis. Additionally, CC (5 and 10 μM) inhibited meiosis (p < 0.05) in CEO and DO cultured for 7 h. Okadaic acid (1 μM) reversed the inhibitory effect of MET (2 mM) and CC (5 μM; p < 0.05) but not of AICAR (1 mM). Phosphorylation of the alpha subunit of AMPK on Thr172 is required for activation. Based on western blot analysis, AICAR, MET and CC did not affect Thr172 phosphorylation levels in DO and oocytes from complexes (p > 0.05). In cumulus cells, Thr172 phosphorylation decreased after 3 h of culture (p < 0.05), regardless of the presence of AMPK modulators in the culture medium. Higher concentrations of AICAR (2 mM) and MET (10 mM) did not affect Thr172 phosphorylation, but phosphorylation on Ser79 of ACC, a substrate of AMPK, was increased in response to MET (p < 0.05). In conclusion, we inferred that the inhibitory effect of AICAR and MET on bovine oocyte meiosis was probably not mediated through activation of AMPK. Moreover, these compounds probably inhibited meiosis through different pathways.

Aiming to improve the developmental competence of bovine oocytes during meiotic block, this study evaluated the effects of a serum replacer (Knockout SR®) and hormones (gonadotropins and estradiol) supplementation of prematuration medium (TCM119 with 0.5 mM IBMX [IBMX group] or 25 μM roscovitine [ROSC group]) on the kinetics of oocyte nuclear maturation and embryo development. Most IBMX and ROSC oocytes prematured for 8 h culture remained in the GV stage (70.3% and 73.1%, respectively; p > 0.05) similar to Control 8 h (63.5%) and to control immature oocytes (Control 0 h, 92.5%). After prematuration for 16 h, no oocytes remained in the GV stage at similar rates to those recently aspirated (p < 0.05); GV rates in ROSC (32.4%) were higher (p < 0.05) than in the Control 16 h group (8.6%), but similar (p > 0.05) to IBMX (9.7%). After in vitro maturation (IMV) for 24 h, metaphase II (MII) rates for oocytes prematured during 8 h were similar (p > 0.05) between control and treatments (65.0–71.7%). Similarly, MII rates oocytes prematured during 16 h were similar (p > 0.05) between all groups (45.9–60.4%). Cleavage rates (67.8–78.2%), embryonic development in day-7 (25.0–35.6%) and hatching rates in day-8 (2.5–11.3%) oocytes blocked during 8 h were similar for all groups (p > 0.05). Results indicate that addition of Knockout SR® and hormones to meiotic block culture with IBMX and roscovitine negatively affected meiotic arrest, but did not impair oocyte nuclear maturation and acquisition of developmental competence.

Rat hepatoma Reuber H-35 cells produce a unique compound designated as Fr.B-25, a 2-cell stage-specific inhibitor of the cleavage of preimplantation mouse embryos cultured in vitro. Here, we identified Fr.B-25 as a purine nucleoside, 5′-deoxy-5′-methylthioadenosine (MTA), by mass spectroscopic analysis. All of the biological activities examined of authentic MTA on the development of mouse zygotes were indistinguishable from those of Fr.B-25. The mechanism of MTA action in the development of preimplantation mouse embryos was probably different from those of hypoxanthine and adenosine, which are well-characterized purine nucleosides that act as inhibitors of the cleavage of mouse 2-cell embryos. From the shared molecular and biological properties of Fr.B-25 and MTA, we concluded that Fr.B-25 is MTA. To the best of our knowledge, this is the first delineation of the effect of MTA on the development of preimplantation mammalian embryos cultured in vitro.

Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus–oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.

The effects of specific components in culture medium on embryo physiology have been extensively investigated to optimize in vitro culture systems; however, little attention has been paid to antibiotics, the reagents used most commonly in culture systems to prevent contamination. To investigate the potential effects of routine use of antibiotics on cultured embryos, mouse zygotes were cultured with or without antibiotics. In both groups, the developmental rate and cell number of blastocysts appear to be normal. The proportion of embryos with blastomere fragmentation increased slightly when embryos were cultured with antibiotics. In contrast, the presence of antibiotics in the embryo culture system significantly disturbs expression of zygotically activated genes, damages chromatin integrity and increases apoptosis of cultured embryos. These results provide evidence that, when cultured with antibiotics, embryos with normal appearance may possess intrinsic physiological and genetic abnormalities. We demonstrate that the adverse effects of antibiotics on mammalian embryos are more severe than we previously presumed and that antibiotics are not essential for sterility of embryo culture system therefore abolishing antibiotic supplementation during embryo culture.

Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

p27BBP/eIF6 (β4 binding protein/eukaryotic initiation factor 6) is a highly conserved protein necessary for cell life. In adult eIF6 mice, a 50% decrease in the protein levels in all tissues is accompanied by a reduction in cell proliferation only in the liver, fat cells and cultured fibroblasts. During X. laevis embryogenesis expression of p27BBP/eIF6 is abundant in high proliferative territories. However, in Xenopus cell proliferation appears unaffected following p27BBP/eIF6 over-expression or down-regulation. Indeed, p27BBP/eIF6 is an anti-apoptotic factor acting upstream of Bcl2 that reduces endogenous apoptosis. We studied p27BBP/eIF6 protein localization in wild type embryos and compared it to proliferation and apoptosis. At the beginning of embryogenesis, high levels of p27BBP/eIF6, proliferation and apoptosis overlap. In later development stages high proliferation levels are present in the same regions where higher p27BBP/eIF6 expression is observed, while apoptosis does not appear specifically concentrated in the same sites. The higher presence of p27BBP/eIF6 would appear related to an increased need of apoptosis control in the regions where cell death is essential for normal development.

Gametogenesis is associated with DNA methylation and involves complicated and delicate gene regulation network in which stem cell marker genes exert their functions. Therefore, it is necessary to investigate DNA methylation profiles of those genes in mature gametes that have an effect on embryo development. However, to date, there are limited data available on these genes in mature gametes of bovine. Here we show methylation profiles in 5′ terminal regions of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) in bovine mature gametes, based on the reasoning that the five genes harbour CpG islands in their own 5′ terminal regions, which are frequently the targets of DNA methylation. The results showed that Oct4 and Fgf4 exhibited significant hypermethylation in sperm compared with that in oocytes (p < 0.01), while Sox2 and Nanog displayed relatively the same methylation levels between sperm and oocytes (p > 0.05). Additionally, Rex1 showed a relatively high methylation level in sperm than in oocytes, although no significant differences were found (p > 0.05). In conclusion, bovine mature gametes exhibited two methylation profiles in terms of the five genes, one being non-sex-specific and the other being sex-specific.

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP3Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP3Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP3Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP3Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP3Rs, probably through a CICR mechanism. Both receptors play a role in Ca2+ release mechanisms although their relative contribution to the activation process is unclear.

The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK–MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK–MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc–PKA, which affects the activity of the Cdc25 phosphatase.