Aurora-A kinase (AURKA), a member of the serine/threonine protein kinase family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little information is known about AURKA in the early cleavage mouse embryo with respect to acentrosomal spindle assembly. In vitro experiments in which AURKA was inactivated with specific inhibitor MLN8237 during the early stages of embryogenesis documented gradual arrest in the cleavage ability of the mouse embryo. In the AURKA-inhibited 1-cell embryos, spindle formation and anaphase onset were delayed and chromosome segregation was defective. AURKA inhibition increased apoptosis during early embryonic development. In conclusion these data suggest that AURKA is essential for the correct chromosome segregation in the first mitosis as a prerequisite for normal later development after first cleavage.

The purpose of this study was to evaluate the effects of FSH and PI3K on the nuclear maturation, viability, steroidogenesis and embryo development of bovine cumulus–oocyte complexes (COCs). Oocyte maturation was achieved with MIV B, MIV B+100 µM LY294002, MIV B+10 ng/mL follicle stimulating hormone (FSH), or MIV B+10 ng/mL FSH+100 µM LY294002 treatments for 22–24 h. After the cultured COCs were denuded, oocytes were separated into those that extruded polar bodies (mature) and those that did not, and real-time polymerase chain reaction (PCR) for BAX, BCL2, LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 genes was performed. The culture medium was collected to determine the levels of 17β-estradiol (E2) and progesterone (P4). The trypan blue test was used to study COC viability, and embryo development was evaluated. FSH increased nuclear maturation and PI3K blocked the maturation but did not influence oocyte viability. BAX and BCL2 expression levels in the cumulus cells were only affected by FSH, and the BAX levels decreased after treatment with LY294002. FSH increased the levels of E2 and P4, however inhibition of PI3K decreased E2 levels. MIV B enhanced levels of LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1, whereas LY294002 inhibited the expression levels of all genes. MIV B+FSH decreased the expression levels of all genes except CYP11A1. LY294002 did not demonstrate any effects in the presence of FSH. Embryo development was significantly decreased when the MIV B+FSH medium was used. In conclusion, FSH controls the steroidogenesis, viability and gene expression in COCs. PI3K plays essential roles in nuclear maturation, steroidogenesis and embryo development.

The role of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations in Bufo arenarum fertilization was analysed. Our results showed that the highest fertilization percentages were obtained when strings of uterine oocytes (UO) were inseminated. Under these conditions, full jelly (FJ), which represents the jelly coats surrounding the oocytes at the time of deposition, contained 68.5 ± 7.0 mM Na+, 27.4 ± 2.4 mM K+, 6.3 ± 0.9 mM Ca2+ and 6.9 ± 0.9 mM Mg2+. When the strings of oocytes were washed in deionized water, these cations diffused into the liquid medium surrounding them. There was a marked similarity between the loss of Ca2+ in the jelly and the decrease in the fertilizability of the UO. Furthermore, the use of chelating agents of divalent cations showed the importance of the Ca2+ contained in the jelly. When Ca2+ was sequestered from the jelly coats by the addition of the chelating agents to the insemination medium as well as by pretreatment of the UO strings, a decrease in fertilization percentages occurred, this effect being dose dependent and more marked with EGTA. These results demonstrate that the Ca2+ in the jelly plays a role in fertilization. Nevertheless, taking into account that during the washing of the jelly other jelly coat components were diffused and considering that the addition of Ca2+ to the insemination medium reverted significantly, but only partially, the loss of fertilizability of jellied UO (washed), the participation of other components in the fertilization mechanism is suggested.

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.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

To determine the ultrastructural changes of sheep cumulus–oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified–warmed sheep COCs.

The earliest stages of development in most animals are under the control of maternally inherited information. The initiation of embryonic gene expression has been reported at the mid-blastula in amphibians and the mid-2-cell stage to the early morula in mammals. In chick embryos, embryonic gene expression was detectable at stage X (morula) and showed marked activation at stage XIII (blastula) with a gradual increase thereafter. Synthesis of rRNA and tRNA was low at stage X and was already the major class of RNA at stage XIII in chick embryos. The observed upregulation of RNA synthesis seems to coincide with a period of extensive fine structural differentiation when the first major cellular migrations start and signal the formation of the primitive streak in the chick embryo.

Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+-selective channel was confirmed. Here, it is reported that the Ca21-selective channel from mouse sperm plasmamembrane displayed a pNa+/Pk+ = 1.6 ± 0.2(n=4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/Pk+ = 2.5±0.3, n=3) with a main open conductance substate of 103 pS and a smaller open substate of 51 PS(600mM K+cis/100 mM Na+trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 ± 0.02, n=3), and a low activity mode (open probability <1%, n=4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 PS (600KCI cis/100KCI trans), was identified. It displayed a high, nearly constant open probability (∼0.90)in the 0 to –80 mV range.

The topic of monozygotic twinning (MT) on which the recent paper by Herranz (Herranz, 2013) focuses is indeed a fascinating one, not only for the embryologist. And, as pointed out by the author, it may on principle also be relevant for ethical discussions in the context of embryo research and stem cell work in humans. Obviously, this is the reason why the paper was published in Zygote, in spite of the fact that it concentrates on history and speculation. When reading through the paper, however, I have ended up disappointed.

The effects of two demethylating drugs with a different mechanism of action (5-azacytidine (Aza) and L-ethionine (Eth)) on mouse preimplantation development were investigated. Preimplantation embryos were cultured for 24 h in the presence of the drug and for an additional 24 or 48 h (depending on the cleavage stage) in medium supplemented with bromodeoxyuridine to reveal sister chromatid exchanges (SCEs) and the number of cell cycles performed before harvesting. Striking differences between the two drugs were observed in their influence on proliferation of blastomeres, primary differentiation and sister chromatid differentiation (SCD), and in the pattern of DNA methylation and the frequency of SCEs per cell. At a final concentration of 1 μM Aza had no effects, whereas higher concentrations stopped development of all stages except the zygote. In contrast Eth treatments (5 mM) resulted in a severe reduction of the mean cell number per embryo in comparison with controls. Moreover both the absence of blastocyst formation and no effects on mitotic activity were detected. The most prominent effect of Eth was detected at the zygote and 4-cell stages. An unexpected decrease in SCE frequency in Eth-treated morulae and 4-cell embryos has been observed. Data are explained taking into account the different mechanisms of action of the agents.

This study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin’s solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

This study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal–Wallis test with a P-value <0.05 considered to be significant. Approximately 87.8% of the oocytes remained at germinal vesicle stage (GV) after 6 h of PM with 5 μM of cilostamide, confirming the meiotic block. Embryo development in IVP was similar (P > 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.

This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 μm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.

The zona pellucida (ZP) is an extracellular glycoprotein coat that is deposited around the oocyte during folliculogenesis and performs several functions that relate to fertilisation and preimplantion development. In eutherian mammals it consists of three major glycoproteins – ZPA, ZPB, and ZPC – but little is known about its molecular constitution in marsupials. We have isolated the cDNA encoding the ZPA homologue in two distantly related marsupial series: the possum, Trichosurus vulpecula (a phalangerid) and the dunnart Sminthopsis crassicaudata (a dasyurid). The two cDNA sequences were 86% identical and showed extensive regions of homology to eutherian ZPA proteins, particularly in the central region of the molecule. Many other features of the ZPA protein, except the positioning of the N-linked glycosylation sites, were also conserved between marsupials and eutherians. ZPA expression was shown to occur maximally in the cytoplasm of the oocyte primary follicles with a little, but significant, expression in oocytes of both primordial follicles and in the cytoplasm of the oocyte in follicles with an antral cavity. No expression was seen in surrounding follicle or granulosa cells

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose . Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 μM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.

The investigation presented in this paper was conducted on the effect of oocytes activation on frozen–thawed human immature oocytes followed by in vitro maturation (IVM). A total of 386 failed-matured oocytes (germinal vesicle (GV) and metaphase I (MI) stages) was randomly divided into two groups: fresh group and vitrification group, GV group and MI group, respectively). The matured oocytes were subject to intracytoplasmic sperm injection (ICSI) after IVM had been carried out. The vitrification group was randomly divided into two groups: controlled and artificial oocyte activation (AOA). The injected oocytes in the controlled group were cultured in cleavage medium. The AOA group oocytes were activated by exposing them to 7% anhydrous alcohol for 6 min then cultured in cleavage medium as well. The rates of fertilization and early embryonic development were compared between the controlled and AOA groups. In MI vitrification group, the high-quality embryo formation rate and blastocyst formation rate were significantly higher in the AOA group than in the controlled group (P < 0.01). In the GV vitrification group, the high-quality embryo formation rate was significantly higher in the AOA group than in the controlled group (P < 0.05). These results indicate that AOA may be good for early embryonic development of vitrified immature human oocytes.

We detected that Fas receptor (Fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, Fas ligand (FasL) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. These results were supported by an indirect immunofluorescence (IIF) test with anti-Fas monoclonal antibody (mAb)/FasL Ab. The lysates from granulosa cells were precipitated by anti-FasL Ab, exhibiting a specific band at 40 kDa. When zona pellucida (ZP)-free eggs were incubated with granulosa cells in vitro, apoptosis (DNA fragmentation) was induced in the eggs, as detected by the terminal deoxynucleotide transferase mediated dUTP-nick end labelling (TUNEL) method. To examine whether FasL-bearing cells can induce apoptosis in eggs expressing Fas through molecular interaction between FasL and Fas, we employed a baculovirus expression system to generate FasL on the surface of Spodoptera frugiperda (Sf9) cells. The co-incubation of eggs with Autographa californica nuclear polyhedrosis virus (AcNPV) and FasL transfected Sf9 (Sf9–FasL) cells in vitro was performed and resulted in the induction of apoptosis in eggs as detected by the TUNEL method. Apoptosis was absent in eggs co-incubated with AcNPV-1393 transfected Sf9 (Sf9−1393) cells. Thus, ovarian atresia was suggested to be induced by molecular interaction between FasL on granulosa cells and Fas on oocytes/eggs during oogenesis.

To examine the possibility that epidermal growth factor (EGF) and its receptor (EGF-R) are expressed throughout folliculogenesis, we studied the presence and distribution of EGF and EGF-R in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of proteins, or used for the isolation of follicles, luteal cells and ovarian surface epithelium to study mRNA expression for EGF and EGF-R, using the reverse transcriptase polymerase chain reaction. EGF protein and mRNA were found in primordial, primary and secondary follicles as well as in small and large antral follicles and in surface epithelium, but in corpora lutea only the protein could be detected. Antral follicles expressed EGF mRNA in oocyte, cumulus, mural granulosa and theca cells. For EGF-R, both protein and mRNA were present at all stages of follicular development and in all antral follicular compartments. EGF-R protein and mRNA were also found in corpora lutea and surface epithelium. It is concluded that EGF and its receptor are expressed in goat ovarian follicles at all stages of follicle development, in corpora lutea, and in ovarian surface epithelium.

The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.