Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes

Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.     

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12–22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.     

Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments

Studies were conducted to compare viability of immature and mature porcine oocytes vitrified in ethylene glycol (EG) using open-pulled straws (OPS). Oocytes that had been allowed to mature for 12 h (germinal vesicle group; GV) and 40 h (metaphase II group; MII) were divided into three treatments: (1) control; (2) treated with cytochalasin B and exposed to EG; and (3) treated with cytochalasin B and vitrified by stepwise exposure to EG in OPS. After warming, a sample of oocytes was fixed and evaluated by specific fluorescent probes before visualization using confocal microscopy. The remaining oocytes were fertilized and cleavage rate was recorded. Exposure of GV oocytes to EG or vitrification had a dramatic effect on spindle and chromosome configurations and no cleavage was obtained after in vitro fertilization. When MII oocytes were exposed to EG or were vitrified, 18 and 11% of oocytes, respectively, maintained the spindle structure and either EG exposure or vitrification resulted in substantial disruption in microfilament organization. The cleavage rates of mature oocytes after being exposed to EG or after vitrification were similar (14 and 13%, respectively) but were significantly less than that of control oocytes (69%). These results indicate that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII porcine oocytes had better resistance to cryopreservation than GV stage 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.     

Inhibition of phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase leads to suppression of p34(cdc2) kinase activity and meiotic progression beyond the meiosis I stage in porcine oocytes surrounded with cumulus cells

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 microM LY294002 or 10 microM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34(cdc2) kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34(cdc2) kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.     

Use of polarized light microscopy in porcine reproductive technologies

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) could be useful for a non-invasive evaluation of the meiotic spindle and may allow removal of nuclear structures without fluorochrome staining and ultraviolet exposure. In this study, PLM was used to assess its potential application in porcine reproductive technologies. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine its effects on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM; and to assess the efficiency oocyte enucleation assisted with PLM. In the first experiment, the presence of microtubule-polymerized protein was assessed and confirmed in oocytes (n = 117) by immunostaining and chromatin detection. In the second experiment, oocytes (n = 160) were exposed or not (controls) to PLM for 10 minutes, and then parthenogenetically activated and cultured in vitro. In the third experiment, development competence of oocytes with a positive or negative signal to PLM was analyzed after in vitro fertilization. Finally, oocytes (n = 54) were enucleated using PLM as a tool to remove the meiotic spindle. A positive PLM signal was detected in 98.2 % of the oocytes, which strongly correlated (r = 1; p < 0.0001) with the presence of microtubule-polymerized protein as confirmed by immunostaining. Oocytes exposed to PLM did not differ significantly from controls on cleavage, total blastocyst, expanded blastocyst rates and total cell numbers. The percentage of oocytes at the MII stage and blastocyst formation rate in the negative PLM group significantly differed from control and PLM positive groups. Overall efficiency of spindle removal using the PLM-Oosight system was 92.6%. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured porcine oocytes and does not exert detrimental effects on porcine oocyte developmental competence. Selecting oocytes by the presence of a PLM signal provides limited improvement on IVF results. Finally, PLM appears as an efficient method to enucleate porcine oocytes.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

Pig oocyte vitrification by cryotop method: effects on viability, spindle and chromosome configuration and in vitro fertilization

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.     

Ultra-structural changes and developmental potential of porcine oocytes following vitrification

This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P<0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P<0.05) in activated group than the group without activation. Significant differences (P<0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation

Various pathological stimuli such as radiation, environmental toxicants, oxidative stress, and heat shock can initiate apoptosis in mammalian oocytes. Experiments were performed to examine whether apoptosis mediated by group II caspases is the cause for disruption of oocyte function by heat shock applied during maturation in cattle. Bovine cumulus-oocyte complexes (COCs) were cultured at 38.5, 40, or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were at 38.5 degrees C and 5% (v/v) CO2 for all treatments. In the first experiment, exposure of COCs to thermal stress during the first 12 h of maturation reduced cleavage rate and the number of oocytes developing to the blastocyst stage. In the second experiment, a higher percentage of TUNEL-positive oocytes was noted at the end of maturation for oocytes matured at 40 and 41 degrees C than for those at 38.5 degrees C. In addition, the distribution of oocytes classified as having high (>25 intensity units), medium (15-25 intensity units), and low (<15 intensity units) caspase activity was affected by treatment, with a greater proportion of heat-shocked oocytes having medium or high activity. In the third experiment, COCs were placed in maturation medium with vehicle (0.5% [v/v] DMSO) or 200 nM z-DEVD-fmk, an inhibitor of group II caspases. The COCs were matured at 38.5 or 41 degrees C, fertilized and cultured for 8 days. The inhibitor blocked the effect of heat shock on cleavage rate and the percentage of oocytes and cleaved embryos developing to the blastocyst stage. In conclusion, heat shock during oocyte maturation can promote an apoptotic response mediated by group II caspases, which, in turn, leads to disruption of the oocyte's capacity to support early embryonic development following fertilization.     

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 μm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.     

Utility of rapidly matured oocytes as recipients for production of cloned embryos from somatic cells in the pig

The present study was conducted to examine the utility of rapidly matured oocytes as recipients for production of porcine embryos reconstituted with adult skin fibroblasts and whether arrest of meiotic resumption of recipient oocytes at the germinal vesicle (GV) stage by dibutyryl cyclic AMP (dbcAMP) improves in vitro developmental rates after reconstruction. At 24 h of maturation in the medium, 36.3% of oocytes reached the metaphase II (MII) stage. At 30 h of maturation, the percentage (71.4%) of MII oocytes did not significantly differ from that (78.0%) at 42 h of maturation. When MII oocytes recovered at 24 h of maturation were used as recipients, 22/156 (14.1%) cloned embryos developing to the blastocyst stage was significantly (P < 0.05) higher than those of embryos reconstituted with oocytes collected at 30 h (5/168; 3.0%) and 42 h (13/217; 6.0%) of maturation. Culture of oocytes in medium containing 1 mM dbcAMP for 20 h maintained 72.9% in the GV stage, whereas only 15.0% of nontreated oocytes were in the GV stage (P < 0.05). The effect of dbcAMP was reversible. However, the treatment of recipient oocytes with dbcAMP did not affect the development of reconstructed embryos when compared with nontreated oocytes. These results indicate that rapidly matured oocytes are superior in their ability to support development of porcine reconstructed embryos; however, arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP does not improve reconstructed embryo developmental rates.     

Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method

This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 microg/mL, 0.05 microg/mL or 0.1 microg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 microg/mL. When oocytes were microinjected on a warm stage at 30 degrees C, the percentage of EGFP-expressing embryos was higher than that at 38.5 degrees C (40.1% vs. 20.9%, P<0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P<0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P<0.01). However, neither incubation temperatures of spermatozoa and DNA (4 degrees C, 24 degrees C or 39 degrees C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.     

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.     

Effect of 1,2-propanediol versus 1,2-ethanediol on subsequent oocyte maturation, spindle integrity, fertilization, and embryo development in vitro in the domestic cat

This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature cat oocyte as a prerequisite to formulating a successful cryopreservation protocol. In experiment 1, immature oocytes were exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25 degrees C) or 0 degrees C for 30 min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure to 3.0 M PrOH at 0 degrees C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control, except 1.5 M PrOH at 25 degrees C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25 degrees C) or not (1.5 M PrOH at 25 degrees C). After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to CPA except after exposure to 1.5 M PrOH at 25 degrees C, a treatment that still allowed approximately 60% of the oocytes to reach MII and approximately 20% to form blastocysts.     

Assessment of canine oocyte viability after transportation and storage under different conditions

To improve assisted reproductive technologies in the domestic dog, different transport treatments were evaluated for their ability to maintain viability of canine oocytes, as assessed by esterase activity 8h after storage or after 48 h of in vitro maturation (IVM) culture. In Experiment 1, ovaries were transported within reproductive tracts or were excised and stored at either 20 or 37 degrees C in phosphate buffered saline. Oocytes collected from reproductive tracts transported at 37 degrees C had the greatest viability after storage (P<0.05). However, after IVM there were no significant differences among any of the four storage conditions in oocyte viability or meiotic resumption (P=0.05). In Experiment 2, isolated oocytes were transported in either TCM-199 with Hank's salts and Hepes buffer or in TL-Hepes at either 20 or 37 degrees C, or in maturation medium equilibrated with 5% CO(2) at 37 degrees C. In Experiment 2, oocytes transported in Hepes buffered media at 37 degrees C had greater viability rates after storage than did those transported in these same media at 20 degrees C or in sodium bicarbonate buffered medium at 37 degrees C (P<0.001). After IVM, oocytes transported in the 37 degrees C treatment groups had greater viability rates than did those transported at 20 degrees C (P<0.01). Overall, isolated oocytes transported at 37 degrees C had greater rates of meiotic resumption than did those transported at 20 degrees C (P<0.05). Taken together, these data indicate that canine oocytes exhibited sensitivity to lesser temperatures and maintained greater rates of viability during transport at 37 degrees C. Isolated oocytes maintained greater viability than oocytes transported in situ. Hepes buffered media increased viability rates for isolated oocytes transported at 37 degrees C compared to a similar medium buffered with sodium bicarbonate.     

Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5 degrees C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5 degrees C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41 degrees C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.     

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)