The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 μM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 μM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 μM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.     

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.     

Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes

This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 μM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture. Mol. Reprod. Dev. 51:339–345, 1998. © 1998 Wiley-Liss, Inc.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Cumulus cells accelerate aging of mouse oocytes

The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.     

Cumulus cells accelerate aging of mouse oocytes by secreting a soluble factor(s)

Control of oocyte aging in vitro is important for both human-assisted reproduction and animal embryo technologies because fertilization or artificial activation of aged oocytes results in abnormal development. Interactions between somatic and germ cells are also an important issue in current biological research. The role of cumulus cells (CCs) in maturation, ovulation, and fertilization of oocytes has been extensively studied, yet little is known about their role in oocyte aging. Although our previous study has shown that CCs accelerate the aging progression of mouse oocytes, the mechanism by which CCs accelerate oocyte aging is unknown. In this study, cumulus-denuded mouse oocytes (DOs) were co-cultured with cumulus-oocyte complexes (COCs) or CC monolayer or cultured in medium conditioned with these cells and changes in the susceptibility to activating stimuli and in MPF activity of oocytes were evaluated after different aging treatments. The results showed that culture with or in medium conditioned with COCs or CC monolayer promoted activation of DOs, indicating that a soluble factor is responsible for the aging-promoting effect. The in vivo and in vitro-matured DOs did not differ in responsiveness to the aging-promoting factor (APF). Heat shock did not accelerate oocyte aging unless in the presence of CCs. The production of APF was not affected by the age or maturation system of COCs, but increased with their density and duration of culture. The results strongly suggest that CCs accelerated oocyte aging by secreting a soluble APF into the medium. Further analysis showed that the APF was heat labile but stable to freezing, it had a threshold effective concentration and can be depleted by DOs.     

Supplementation of maturation medium with CoQ10 enhances developmental competence of ovine oocytes through improvement of mitochondrial function

In vitro maturation (IVM) can impair the balance between antioxidant capacity and oxidative stress, and jeopardize embryo development by increasing oxidative stress, reducing energy metabolism, and causing improper meiotic segregation. Balancing the energy production and reduction of oxidative stress can be achieved by supplementation with coenzyme Q10 (CoQ10), an electron transporter in the mitochondrial inner membrane. To improve the in vitro production of ovine embryos, we studied the effect of CoQ10 supplementation during the maturation of sheep oocytes. A minimum of 100 cumulus‐oocyte complexes (COCs) were matured in the presence of 15, 30, or 50 μM CoQ10 in three to five replicates; next, in vitro fertilization and culture in a subset of oocytes were done. Our data revealed that compared to control oocytes or other concentrations of CoQ10, supplementation with 30 µM CoQ10 resulted in a significant increase in blastocyst formation and hatching rates, improved the distribution, relative mass and potential membrane of mitochondria, decreased the levels of reactive oxygen species and glutathione and lessened the percentage of oocytes with misaligned chromosomes after spindle assembly. The relative expression levels of apoptosis markers CASPASE3 and BAX were significantly reduced in CoQ10‐treated oocytes and cumulus cells whereas the relative expression level of GDF9, an oocyte‐specific growth factor, significantly increased. In conclusion, supplementation with CoQ10 improves the quality of COCs and the subsequent developmental competence of the embryo.     

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM

Di‐(2‐ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine‐disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time‐lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP‐induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP‐exposed oocytes. VB addition during IVM limited DEHP‐induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.     

Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.     

Sperm-derived activating ability does not persist in mouse oocytes inseminated during in vitro maturation

Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by a fusion with metaphase II (MII) oocytes leading to the activation of the hybrids. We have shown, that in the great majority of oocytes inseminated 1-2 hr after germinal vesicle breakdown (GVBD) the sperm-derived activating ability was eliminated. Only few hybrids produced by fusion of MII oocytes with oocytes inseminated during in vitro maturation (M x IVM-P + sperm hybrids) underwent activation, whereas almost all of MII oocyte x zygote hybrids entered interphase. However, frequency of activation of M x IVM-P + sperm hybrids was higher than that of control hybrids, which were obtained by fusion of MII oocytes with oocytes uninseminated during in vitro maturation. Although the difference was not statistically significant, it suggested that in a certain number of oocytes inseminated after GVBD the sperm-derived oocyte-activating factor remained partially active. This was confirmed by our observation that several oocytes, which were inseminated during in vitro maturation and managed to accomplish MII, underwent activation and formed pronuclei when examined 25-26 hr after the beginning of maturation. We have also demonstrated that parthenogenotes, could acquire the sperm-derived activity, as a consequence of sperm injection. MII oocytes were fused with parthenogenotes inseminated by ICSI and all hybrids underwent activation. This result indicated that the ability to induce activation in hybrid, was sperm-derived.     

The ability to achieve meiotic maturation in the dog oocyte is linked to glycolysis and glutamine oxidation

We tested the hypothesis that meiotic competence of dog oocytes is tightly linked with donor follicle size and energy metabolism. Oocytes were recovered from small (<1 mm diameter, n = 327), medium (1–<2 mm, n = 292) or large (≥2 mm, n = 102) follicles, cultured for 0, 24, or 48 hr, and then assessed for glycolysis, glucose oxidation, pyruvate uptake, glutamine oxidation, and nuclear status. More oocytes (P < 0.05) from large follicles (37%) reached the metaphase‐II (MII) stage than from the small group (11%), with the medium‐sized class being intermediate (18%; P > 0.05). Glycolytic rate increased (P < 0.05) as oocytes progressed from the germinal vesicle (GV) to MII stage. After 48 hr of culture, oocytes completing nuclear maturation had higher (P < 0.05) glycolytic rates than those arrested at earlier stages. GV oocytes recovered from large follicle oocytes had higher (P < 0.05) metabolism than those from smaller counterparts at culture onset. MII oocytes from large follicles oxidized more (P < 0.05) glutamine than the same stage gametes recovered from smaller counterparts. In summary, larger‐sized dog follicles contain a more metabolically active oocyte with a greater chance of achieving nuclear maturation in vitro. These findings demonstrate a significant role for energy metabolism in promoting dog oocyte maturation, information that will be useful for improving culture systems for rescuing intraovarian genetic material. Mol. Reprod. Dev. 79: 186–196, 2012. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39°C in a humidified atmosphere with 5% CO₂ in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation. Mol. Reprod. Dev. 49:444–453, 1998. © 1998 Wiley-Liss, Inc.     

The role of mitochondrial transition pores on bovine oocyte competence after heat stress, as determined by effects of cyclosporin A

This study was undertaken to investigate the effects on the nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS) when treating bovine oocytes before in vitro maturation (IVM) with 1 μM cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening. Mitochondrial activity, reactive oxygen species (ROS), and apoptosis levels of the oocytes were also assessed. Nuclear maturation rates of both the HS-exposed oocytes treated with or without CsA groups (HS + CsA or HS group) were significantly lower (P<0.05) than that of the control group, while the rate of the HS + CsA group was significantly higher (P<0.05) than that of the HS group. Furthermore, although the cleavage and blastocyst formation rates of the HS group were significantly lower than those of the control groups (P<0.05), both rates of the HS + CsA group recovered to the same level as those of the control group. The HS group showed a significantly higher ROS level, lower mitochondrial activity in the oocytes, and TUNEL-positive cumulus cells, but not oocytes, compared with those of the control group (P<0.05), whereas the TUNEL-positive and mitochondrial activity levels of the HS + CsA group recovered to those of the control group. These results indicate that 1 μM CsA treatment before IVM may mitigate reduced mitochondrial activity, increase number of apoptotic cumulus cells under HS, and improve the nuclear maturation and developmental competence of bovine oocytes.     

Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes

Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.     

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.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.