Transient increases in the concentration of cytosolic Ca2+ are essential for triggering egg activation events. Increased Ca2+ results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A2 (PLA2) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA2 activator, and arachidonic acid, the main PLA2 reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

We investigated the participation of Gαi protein and of intracellular cAMP levels on spontaneous and progesterone-mediated maturation in Rhinella arenarum fully grown follicles and denuded oocytes.

Although progesterone is the established maturation inducer in amphibians, Rhinella arenarum oocytes obtained during the reproductive period (competent oocytes) resume meiosis with no need for an exogenous hormonal stimulus if deprived of their enveloping follicular cells, a phenomenon called spontaneous maturation. In amphibian oocytes, numerous signalling mechanisms have been involved in the rapid, non-genomic, membrane effects of progesterone, but most of these are not fully understood.

The data presented here demonstrate that activation of the Gαi protein by Mas-7 induced maturation in non-competent oocytes and also an increase in GVBD (germinal vesicle breakdown) in competent oocytes. Similar results were obtained with intact follicles independent of the season. The activation of adenylyl cyclase (AC) by forskolin seems to inhibit both spontaneous and progesterone-induced GVBD. In addition, the high intracellular levels of cAMP caused by activation of AC by forskolin treatment or addition of db-cAMP inhibited maturation that had been induced by Mas-7 and in a dose-dependent manner. Treatment with H-89, a protein kinase A (PKA) inhibitor, was able to trigger GVBD in a dose-dependent manner in non-competent oocytes and increased the percentages of GVBD in oocytes competent to mature spontaneously. The results obtained with whole follicles and denuded oocytes were similar, which suggested that effects on AC and PKA were not mediated by follicle cells. The fact that Mas-7 was able to induce maturation in non-competent oocytes in a similar manner to progesterone and to increase spontaneous maturation suggests that Gαi activation could be an important step in meiosis resumption. Thus, the decrease in cAMP as a result of the regulation of the G proteins on AC and the inactivation of PKA by H-89 could contribute to the activation of MPF (maturation promoting factor) and induce maturation of the oocytes of Rhinella arenarum.

The present study was undertaken to establish an effective method for in vitro maturation (IVM) of denuded oocytes (DOs) by simulating the ovarian three-dimensional status in vivo using buffalo ovarian tissues or cumulus cells, so as to provide a model for investigating the mechanisms of oocyte maturation. Buffalo cumulus–oocyte complexes from ovaries taken at slaughter were denuded by pipetting, and then allocated randomly into four groups for IVM by direct culture in maturation medium (M1, control group), co-culture with a monolayer of cumulus cells (M2), embedded in cumulus cell clumps (M3) and ovarian tissue (M4) for 24 h. The nuclear maturation of DOs was assessed by the extrusion of the first polar body and the cytoplasmic maturation was evaluated by subsequently developmental capacity after parthenogenetic activation. More DOs matured to MII (56.89%) and developed to blastocysts (25.75%) when they were matured in vitro with M3 in comparison with DOs matured in vitro with M1 (45.14 and 15.97%) and M4 (40.48 and 13.49%). Further detection of gap junctions by injecting Lucifer yellow directly into cytoplasm of matured DOs with adherent cumulus cells and scanning with confocal microscope showed that Lucifer yellow were found in nine out of 11 the adherent cumulus cells in M3, indicating that the gap junctions between oocytes and cumulus cells was reconstructed in vitro. These results indicate that co-culture of DOs embedded in cumulus cell clumps can improve their nuclear and cytoplasmic maturation of DOs, possibly through the reconstruction of gap junctions in vitro.

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.

Whale oocytes recovered from follicles can be matured in vitro. Whale sperm and mature oocytes can be used for in vitro fertilization (IVF), and IVF embryos have the ability to develop to morula stage. Whale sperm injected into bovine or mouse oocytes can activate the oocytes and form pronucleus. Interspecies somatic cell nuclear transfer embryos have been reconstructed with whale somatic cell nucleus and enucleated bovine or porcine oocytes, and interspecies cloned embryos can develop in vitro. This paper reviews recent progress in maturation, fertilization and development of whale oocytes.

The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB− (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB+ oocytes than in BCB− (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB+ and BCB− groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB+ vs BCB− (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

Granulosa cells influence the growth and acquisition of the developmental competence of oocytes. We investigated the effects of ageing on the proliferative activity, global genomic DNA methylation, relative telomere length and telomerase activity of bovine granulosa cells. The proliferative activity of cells was examined by bromodeoxyuridine (BrdU) assay, genomic DNA methylation was examined by enzyme-linked immunosorbent assay (ELISA), and relative telomere length and telomerase activity were examined by real-time polymerase chain reaction. We first compared the proliferative activity of the granulosa cells of the medium follicles between in dominant phase ovaries and growth phase ovaries. We observed that the proliferative activity of the granulosa cells of dominant phase ovaries was significantly lower than those of growth phase ovaries. In addition, the proliferative activity of granulosa cells was inversely associated with follicular size. Based on the results, we used granulosa cells harvested from the medium follicles (3–5 mm in diameter) on the surfaces of the dominant phase ovaries collected from cows at a slaughterhouse. The proliferative activity of the granulosa cells harvested from the ovaries of old cows (N = 8; average age 165.1 months) was lower than that of the cells from young cows (N = 8; average age 30.9 months). Global loss of cytosine methylation was detected in the granulosa cells of old cows (N = 12; average age 141.0 months) compared with young cows (N = 15; average age 27.4 months). Although the relative telomere lengths of cumulus cells were similar in the two age groups, the relative telomere lengths and telomerase activity of the granulosa cells from old cows (N = 17 and 9; average age, 164.6 and 151.3 months, respectively) tended to be shorter than those of the cells from young cows (N = 17 and 10; average age 30.6 and 28.1 months, respectively); however, this difference was not significant p = 0.09 and 0.053, respectively). In conclusion, the proliferative activity and genomic global DNA methylation significantly decreased, and the relative telomere lengths and telomerase activity of granulosa cells tended to be shorter with the age of donor cows.

The purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP–EGFP–Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal–Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus–oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days’ culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to primary/secondary follicles in the goat ovaries and that BMP-6 enhances the growth of cultured secondary follicles.

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus–oocyte complexes (COC) were harvested and divided into two groups: G1 – morphologically healthy cells; and G2 – less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

The present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200–250 μm) were isolated and cultured with or without AFs (3–5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus–oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

The present study was conducted to investigate the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) supplemented into porcine maturation medium on reactive oxygen species (ROS) and glutathione (GSH) levels of oocytes, and apoptosis of cumulus cells (CC). In addition, the effect of dbcAMP on embryonic development following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Cumulus–oocyte complexes (COCs) were cultured in 0 mM (control), 0.5 mM, 1 mM, 5 mM, or 10 mM dbcAMP-supplemented medium for 22 h, then for another 22 h without dbcAMP. GSH and ROS levels of oocytes were assessed at 44 h of culture by dichlorohydrofluorescein diacetate or 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin staining, respectively. Additionally, COCs were cultured in 0.5 mM or 1 mM dbcAMP and then fertilized in vitro or activated parthenogenetically. Embryonic development and blastocyst cell numbers and apoptosis levels on day 8 of culture were investigated. CC apoptosis at 44 h of culture and blastocyst apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. GSH levels in the 0.5 mM dbcAMP and control groups were increased (P < 0.05), while levels of oocyte ROS and CC apoptosis in the control, 0.5 mM, and 1 mM dbcAMP groups were significantly lower than the levels in other groups. Cleavage and blastocyst rates, cell numbers, and apoptosis levels were not significantly different in embryos derived by either IVF or PA among the groups, with the exception of significantly increased apoptotic levels in IVF blastocysts produced from oocytes treated with 1 mM dbcAMP. In conclusion, dbcAMP treatment during in vitro maturation (IVM) did not improve embryonic development under our study's parameters compared with control conditions, although 0.5 mM dbcAMP showed significantly higher GSH levels and lower blastocyst apoptotic levels compared with 1 mM dbcAMP.