Studies on chilling sensitivity of early stage zebrafish (Danio rerio) ovarian follicles

Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage ovarian follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish ovarian follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) ovarian follicles, which were chilled in KCl buffer and L-15 medium for up to 144 h at −1 °C in a low temperature bath. Ovarian follicles were also exposed to 2 M methanol or 2 M DMSO in L-15 medium for up to 168 h at −1 and −5 °C, respectively. Control follicles were kept at 28 °C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles. These results were also confirmed following in vitro maturation of the chilled ovarian follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for ovarian follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of ovarian follicles at all stages with methanol being the most effective. The study indicated that stage I and II follicles are less sensitive to chilling than stage III follicles, and that early stage zebrafish ovarian follicles may be better candidates for cryopreservation.     

Transforming growth factor-α in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure

We examined the effects of transforming growth factor-α (TGF-α) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3 mg/mL polyvinyl alcohol (POM) and TGF-α (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-α increased (P < 0.05) the percentage of oocytes that reached metaphase II (24.2%) compared with that of the control (no TGF-α addition; 5.6%). In the presence of gonadotropins, although maturation rate did not differ among TGF-α treatments (75.4% to 84.8%), the rate of blastocyst formation (28.1%) was higher (P < 0.05) in the TGF-α group (28.1%) than that in the control group (15.9%) after in vitro fertilization and embryo culture. An electron microscope study revealed that TGF-α–treated oocytes contained more homogenous lipid droplets than did control oocytes. Moreover, mitochondria surrounded by the endoplasmic reticulum were observed only in the TGF-α–treated oocytes. In blastocysts derived from the latter oocytes, mitochondria with numerous cristae were frequently observed compared with that in blastocysts from control oocytes. When the Day-5 blastocysts obtained from oocytes matured with TGF-α were surgically transferred into four recipients, a total of 29 piglets were farrowed. We concluded that the addition of TGF-α to the defined IVM medium of porcine oocytes improved the subsequent blastocyst formation and that the blastocysts produced by the defined in vitro production system have developmental competence to full term after embryo transfer.     

Effect of progesterone and/or estradiol treatments prior to induction of ovulation on subsequent luteal lifespan in anestrous Nelore cows

Oocyte quality is the main factor that determines blastocyst yield; any factor that could affect it, such as apoptosis, could impair subsequent embryonic development. Our aim was to investigate the incidence of apoptosis in prepubertal goat oocytes and cumulus cells, assessed by Annexin-V staining and TUNEL assay, and their effect on embryo development. Oocyte-cumulus complexes (COCs) from slaughtered females were collected and classified depending on COC morphology as: Healthy (H) and Early Atretic (EA). Each one of these groups was classified depending on oocyte diameter: A: 110–125 μm, B: 125–135 μm and C: >135 μm. The COCs were IVM for 27 h, IVF with fresh semen and IVC for 8 days after insemination. Apoptosis analyses were performed before and after maturation. Annexin-positive oocytes decreased with diameter in the EA class (immature oocytes: A: 42.6%; B: 30.3%; C: 21%; IVM-oocytes: A: 17.5%; B: 4.8%; C: 0%), while TUNEL assay showed a decrease of apoptosis in the largest oocytes before and after IVM only in Healthy oocytes (immature oocytes: A: 51.5%; B: 43.3%; C: 12.1%; IVM-oocytes: A: 31.7%; B: 12%; C: 0%). Blastocyst rate increased with increasing oocyte diameter, and it was higher in H than in EA oocytes (Healthy; A: 0%; B: 5.3%; C: 14.4%; Early atretic: A: 0.3%; B: 4.1%; C: 5.1%). Oocyte diameter and COC morphology had no effect on the percentage of apoptosis in blastocyst cells. In conclusion, oocyte developmental competence in prepubertal goats is influenced by oocyte diameter and COC morphology.     

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature andin vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20°C, while a very broad phase transition, which centered around 10°C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13°C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4°C (51%) or holding them at 38°C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed whenin vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4°C, and 26% underwent maturation and 19% underwent fertilizationin vitro. In vitro-matured oocytes that were cooled to 4°C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused within vitro-matured and enucleated oocytes, and then cooled to 4°C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca²⁺ chelator and mitochondrial Ca²⁺ uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P < 0.05) than those in the other vitrified groups (10.3–12.3% and 6.2–10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7–21.6% and 8.5–155%, respectively). When BAPTA-AM, an intracellular Ca²⁺ chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5–36.2% and 25.0–26.9%, respectively) were significantly higher (P < 0.05) than those in the absent vitrified groups (17.2–24.2% and 12.9–19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca²⁺ uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8–38.4% and 14.4–24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization.These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.     

Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 °C temperature

Temporal storage of ovaries can provide opportunity to rescue oocytes from ovaries of endangered felids. The objective of the study was to examine the effect of different storage periods (2, 24 and 48 h) of ovaries at 4 °C for maturation of cat oocytes in vitro. Ovaries were collected from 25 domestic cats at various stages of the estrous cycle by routine ovariohysterectomy following anesthesia at different local veterinary clinics, and maintained in physiological saline at 4 °C for 2, 24 or 48 h until oocytes recovery. Selected COCs were maturated at 38 °C for 48 h in four-well petri dishes, which included 500 μL modified synthetic oviduct fluid (mSOF) medium under mineral oil in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere incubator. After the in vitro maturation period, there were no differences between the rate of oocytes matured at MII stages in 2 and 24 h storage groups (50.7% and 48.2% respectively, p > 0.05). However, the same result for the 48 h group was significantly lower than the 2 and 24 h groups (28.0%, p < 0.001).Our results suggest that while 2 or 24 h storage of ovaries at 4 °C does not affect the meiotic competence of oocytes in vitro, 48 h storage of ovaries decrease the results dramatically.     

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca²⁺ chelator and mitochondrial Ca²⁺ uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P < 0.05) than those in the other vitrified groups (10.3–12.3% and 6.2–10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7–21.6% and 8.5–155%, respectively). When BAPTA-AM, an intracellular Ca²⁺ chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5–36.2% and 25.0–26.9%, respectively) were significantly higher (P < 0.05) than those in the absent vitrified groups (17.2–24.2% and 12.9–19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca²⁺ uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8–38.4% and 14.4–24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization.These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.     

The kinase inhibitor indirubin-3'-oxime prevents germinal vesicle breakdown and reduces parthenogenetic development of pig oocytes

Oocytes undergo spontaneous germinal vesicle breakdown (GVBD) after being released from the follicular environment; this potentially prevents manipulation of the oocyte at the germinal vesicle (GV) stage. The objectives of this study were to investigate the effects of indirubin, a potent cdc2 kinase inhibitor, on GVBD and microtubular structure of porcine oocytes. Cumulus-oocyte-complexes (COCs) were collected from abattoir-derived ovaries and were randomly allocated to different concentrations of indirubin treatments (0, 10, 25, 50, and 100 microM in Experiment 1 and 0, 50, 75, and 100 microM in Experiment 2) during 44 h of IVM. The influences on the GVBD, microtubules, and maturation rates were evaluated using epifluorescence microscopy. The percentages of oocytes remaining at the GV stage were 0, 16, 26, 69, and 85% for oocytes treated with 0, 10, 25, 50, and 100 microM of indirubin, respectively, which differed among treatment groups (P<0.05). However, there were no significant differences between the oocytes treated with 75 and 100 microM (79 and 81%). The cytoplasmic microtubules were fragmented in oocytes maintained at the GV stage and the chromatin became condensed or aggregated. When COCs were incubated with indirubin (50-75 microM) for 22 h and then transferred to maturation medium for 44 h (Experiments 3-5), the percentages of oocytes reaching the metaphase II stage were generally higher than when the COCs were cultured in the presence of the drug for 44 h (62-65% versus 44-46%). However, the parthenogenetic development of the oocytes in Experiment 6 was reduced significantly in drug-treated oocytes. In summary, treatment with 50-75 microM of indirubin effectively prevented GVBD in porcine oocytes, but the developmental competence of the oocytes was compromised.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes

The aim of this study was to compare the effectiveness of two different vitrification carrier systems for oocyte cryopreservation. In vitro matured (IVM) bovine oocytes were vitrified in open pulled straws (OPS) or flexipet denuding pipettes (FDP), and the effects of cryopreservation determined on the cytoskeletal components and developmental capacity of the oocytes. Three experimental groups were established according to whether the oocytes were vitrified in OPS (OPS group), FDP (FDP group) or left untreated (CTR group). Twenty two hours after the onset of maturation, sub-groups of 2–4 oocytes were pre-equilibrated in 1 mL of Hepes-TCM 199 with 20% fetal calf serum (FCS) (HM), 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 30 s. The oocytes were then transferred to a 20-μL drop of HM containing 20% DMSO, 20% EG and 0.5 M of sucrose, which was used to load the OPS or FDP before their immersion in liquid nitrogen (LN₂). Oocytes were thawed by plunging the OPS or FDP into 0.25 M sucrose in HM, and then placed for 5 min each in 0.15 and 0 M sucrose in HM. After warming, spindle configuration, chromosome distribution and embryo development were assessed. Frozen–thawed semen was used for fertilization. Zygotes were denuded at 22 h post-insemination, and cultured in SOF medium for 9 days at 38.5 °C in a 5% CO₂, 5% O₂ and 90% N₂ atmosphere. All experiments were performed using both cow and calf oocytes to establish sensitivity differences. After in vitro fertilization and culture, oocytes in the FDP group showed a lower cleavage rate than those in the OPS or control groups (P < 0.05), while blastocysts were only obtained in the OPS group, at a lower rate than controls. After warming, double fluorescent staining revealed higher rates of spindle and chromosome abnormalities in the FDP group compared to the OPS group (P < 0.05). No differences between cow and calf oocytes were observed in the different experiments. Our results indicate that the carrier system affects the capacity of IVM oocytes to survive cryopreservation. Unexpectedly, the flexipet denuding pipette failed to improve results and high rates of clustered chromatin and abnormal spindles were observed in calf and cow oocytes vitrified by the FDP method. In conclusion, the use of the flexipet denuding pipette modifies the cytoskeletal components and compromises the developmental capacity of in vitro matured calf and cow oocytes.     

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

Phosphatidylinositol-3-kinases (PI3Ks) play pivotal roles in meiotic progression of oocytes from metaphase I to metaphase II stage. Using a Class III-specific inhibitor of PI3K, 3-methyladenine (3MA), this study shows that Class III PI3K may be essential for meiotic progression of porcine oocytes beyond germinal vesicle (GV) stage. Treatment of immature porcine oocytes with 3MA for 22-42 h arrested them at the GV stage, irrespective of the presence or absence of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). The GV-arresting effect of 3MA was, however, completely reversible upon their further culture in the absence of 3MA for 22 h. When cumulus-oophorus-complexes (COCs), arrested at the GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached the MII stage at 42 h of IVM and did not differ from non-treated control oocytes with respect to their ability to fertilize, cleave and form blastocyst (P > 0.05) upon in vitro fertilization (IVF) or parthenogenetic activation (PA). These data suggest that 3MA efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocyte beyond the GV stage.     

Meiotic competence of bovine fetal oocytes following in vitro maturation

The in vitro ability between fetal and cow oocytes to resume meiosis and progression to metaphase-II (M-II) was compared. Cumulus oocyte complexes (COCs) were harvested from 2 to 6 mm follicles from ovaries of 7.5 month to term fetuses and adult cows. Cumulus cells were removed using 3 mg/ml hyaluronidase and repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde, stained with DAPI and evaluated under fluorescent microscopy for nuclear status before in vitro maturation (IVM). COCs from fetal and adult ovaries were also matured in 200 microl droplets of medium 199 supplemented with 10 microg/ml FSH, 10/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM hepes and 10% FBS for 24 h at 39 degrees C and 5% CO(2). Matured oocytes were fixed, stained and evaluated as explained above for nuclear status namely stage of germinal vesicle (GV) development and subsequent meiotic competence. Data were analyzed using chi-square analysis. The majority of fetal oocytes (P<0.05) before IVM were at GV stages GV-I (27.7%), GV-II (37.6%) and GV-V (22.8%) compared to cow oocytes, which were at GV stages IV (28.3%) and V (46.7%). After IVM, fewer fetal oocytes were at earlier stages of GV development and majority (P<0.05) were at GV-V (24.0%), premetaphase (17.4%) and metaphase-I (M-I: 7.2%) stages. However, after IVM, more cow oocytes matured to M-II than did fetal oocytes (93.7% versus 26.9%; P<0.05). In conclusion, fetal oocytes do not mature in vitro as well as cow oocytes. Our findings suggest that the low meiotic competence of fetal oocytes can be attributed to their being at earlier stages of GV development before IVM.     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

Porcine cumulus cell influences ooplasmic mitochondria-lipid distributions, GSH-ATP contents and calcium release pattern after electro-activation

The objective was to explore mechanisms of the influence of porcine cumulus cells (CC) on oocyte maturation. Immature porcine oocytes were matured in groups of denuded oocyte (DOs), cumulus-oocyte complexes (COCs), denuded oocytes co-cultured with CC (DoCC), or with cumulus-oocyte complexes (DoCOCs). Ooplasmic mitochondria-lipid distributions, glutathione (GSH)-adenosine triphosphate (ATP) contents, calcium release pattern, and developmental competence after parthenogenetic activation were assessed after IVM. The portion of matured oocytes after IVM and the developmental competence and GSH content in single oocytes were lower in DOs than in COCs (P < 0.05). In contrast, the maturation rate and development in DoCOCs and COCs were higher than in DoCC and DOs (P < 0.05). The blastocyst rate in DoCOCs was higher than in DOs (P < 0.05), and ATP content in COCs was higher than in all other groups (P < 0.01). In addition, the rate of oocytes with damaged oolemma in DOs (35%) was significantly higher than in COCs (3%), DoCOCs (7%), and DoCC (10%). The rate of oocytes with evenly distributed mitochondria was 70% in DOs, which was significantly lower than in COCs and DoCC (89 and 84%, respectively). The percentage of oocytes with normal lipid droplets distributions in COCs (70%) was significantly higher than in three other groups, whereas both percentages in DoCC and DoCOCs were higher than in DOs (P < 0.05). The duration of [Ca²⁺] rise in DOs was longer than in three other groups, whereas the duration was shortest in COCs. The amplitude of the [Ca²⁺] rise in DOs was significantly lower than in other groups (P < 0.05), but the amplitude did not differ significantly among DoCC, DoCOCs and COCs. In conclusion, the presence of porcine CC during IVM functionally affected ooplasmic mitochondria-lipid distributions and GSH-ATP contents, which may affect the calcium release pattern and developmental competence of oocytes after electro-activation.     

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly (P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher (P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group.In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.     

Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.     

Importance of primary culture conditions for the development of rat ICSI embryos and long-term preservation of freeze-dried sperm

Rat sperm freeze-dried in a solution containing Tris and ethylenediaminetetraacetic acid (EDTA) (TE buffer) can be preserved at 4 °C, and oocytes injected with these sperm developed into offspring though developmental ability was low. We studied the culture conditions to improve the developmental ability of oocytes injected with freeze-dried sperm. After being injected with fresh sperm, the zygotes were cultured in modified Krebs–Ringer bicarbonate (mKRB), modified rat 1-cell embryo culture medium (mR1ECM)/BSA, and mR1ECM with different osmolality, before being cultured in mR1ECM. High proportion of zygotes cultured in mKRB (270 mOsm) before being cultured in mR1ECM developed into blastocysts compared to zygotes cultured only with mR1ECM (50% vs. 28%, P < 0.05). Culturing in mKRB also led to a high proportion of zygotes developing into blastocysts after the injection of freeze-dried sperm than zygotes cultured only with mR1ECM (32% vs. 15%, P < 0.05). Offspring (16%) were obtained when 19 2-cell embryos derived from oocytes that had been injected with freeze-dried sperm preserved at 4 °C for 1 year were transferred. This study demonstrated that the culture conditions soon after the injection of sperm markedly influenced the subsequent development of embryos. Also, rat sperm after freeze-drying in TE buffer were preserved at 4 °C for long term without their deterioration.