Caffeine supplementation during IVM improves frequencies of nuclear maturation and preimplantation development of dromedary camel oocytes following IVF

Caffeine supplementation during oocyte IVM has been reported to improve preimplantation embryo development and the quality of in vitro–produced blastocysts in a range of species; but no studies have been done in camels. The present study investigated the effect of caffeine supplementation during dromedary camel oocyte IVM on nuclear maturation rates, IVF events, and subsequent preimplantation development. Cumulus–oocyte complexes obtained at slaughter were matured in vitro in caffeine supplemented medium either for 30 hours (caffeine 30 hours) or in the medium without caffeine supplement for 24 hours and then transferred to freshly prepared IVM medium supplemented with 10 mM caffeine for another 6 hours (caffeine 6 hours). Cumulus–oocyte complexes matured for 30 hours in the medium without caffeine supplement were used as a control. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels collected from a local slaughterhouse. Eighteen hours after insemination, presumptive zygotes were cultured in modified KSOMaa medium for 7 days. Maturation and fertilization rates were significantly higher in the caffeine 6-hour group compared with the control group (P < 0.05), whereas IVM of oocytes in caffeine-supplemented medium for 30 hours did not affect these parameters (P > 0.05). Interestingly, IVM of oocytes in caffeine supplemented medium for 6 hours significantly (P < 0.05) increased the frequencies of blastocyst development by more than two-fold when compared with control (27.78% vs. 11.76%). In conclusion, culturing dromedary camel oocytes in maturation medium without caffeine for 24 hours and then in the medium supplemented with 10 mM caffeine for 6 hours during 30-hour IVM can significantly improve frequencies of nuclear maturation, fertilization rate, and subsequent preimplantation development.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

Cumulus cells during in vitro fertilization and oocyte vitrification in sheep: Remove,maintain or add?

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.     

Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate,ultrastructure, and mitochondrial DNA copy number

Viable lambs can be produced after transfer of in vitro–derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine–treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0.089) and an altered distribution of lipid droplets (P = 0.076). In conclusion, acetyl-L-carnitine can be used to increase the in vitro blastocyst rate of juvenile oocytes and therefore to improve juvenile in vitro embryo transfer methods. These methods can be used for livestock improvement by increasing the rate of genetic gain. Further work is required to identify the contents of the vesicles and confirm the mode of action of acetyl-L-carnitine in improving oocyte competence.     

Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles

Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5–1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P < 0.05) than that observed after 14 days of culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P < 0.05) when 12 or 14 days of IVG culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P < 0.05) when pre-IVM culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P < 0.05) after 12 days. Blastocyst production rate for oocytes obtained after 12 days of IVG culture (24.5%) was greater (P < 0.05) than for oocytes obtained after 14 days (9.9%). In conclusion, 12 days IVG followed by pre-IVM culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.     

Effects of vitrification for germinal vesicle and metaphase II oocytes on subsequent centromere cohesion and chromosome aneuploidy in mice

The present study examined the effect of vitrification on oocyte aneuploidy and centromere cohesion. Firstly, germinal vesicle (GV) and in vitro matured oocytes (metaphase II, MII) were vitrified by open-pulled straw method. Secondly, thawed GV oocytes were matured in vitro to detect the aneuploidy rate and the sister inter-kinetochore (iKT) distance (in situ spreading and immunofluorescent staining). The results revealed that the sister iKT distance and the aneuploidy rate in eggs matured from vitrified-thawed GV oocytes were higher than that from in vivo matured, in vitro matured, and in vitro matured frozen oocytes (0.47 ± 0.03 vs. 0.33 ± 0.01 vs. 0.33 ± 0.02 vs. 0.34 ± 0.01 μm; P < 0.01 and 22.9% vs. 6.5% vs. 5.8% vs. 11.8%; P < 0.05, respectively). Furthermore, the percentage of sister chromosome pairs whose sister iKT distances were higher than 0.9 μm in eggs matured from vitrified-thawed GV oocytes (8.7%) was higher than that from in vivo matured (1.6%), in vitro matured (1.6%), and in vitro matured frozen oocytes (2.3%) (P < 0.05). The sister iKT distance was associated with centromere cohesion. To investigate whether vitrification of GV oocytes deteriorated centromere cohesion by affecting cohesin complex formation, thawed and fresh GV oocytes were used to detect the cohesin subunits (SMC1β, STAG3, SMC3, and REC8) mRNA expression (quantitative real-time polymerase chain reaction). The relative expression of three cohesin subunits (SMC1β, STAG3, and SMC3) was significantly decreased in GV oocytes after vitrification. In conclusion, vitrification of GV oocytes may result in the subsequent deterioration of centromere cohesion and an increase in the aneuploidy rate. MII oocytes may be the ideal candidate to avoid aneuploidy for fertility cryopreservation.     

Embryo development after vitrification of immature and in vitro-matured equine oocytes

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO₂ or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO₂- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO₂ (20.3 ± 4.6%). Cleavage for MAT CR- CO₂ and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO₂), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Soil chromium bioremediation: Synergic activity of actinobacteria and plants

The aim of this work was to evaluate a strategy to reduce the bioavailable chromium fraction in soil, using a Cr(VI) resistant microorganism, Streptomyces sp. MC1, under non sterile conditions, with maize plants as bioindicator and/or bioremediator.Soil samples were contaminated with 100, 200 and 400 mg kg⁻¹ of Cr(VI) or Cr(III). Bioavailable chromium (35%) was only detected in samples with Cr(VI). Soil samples with Cr(VI) 200 mg kg⁻¹ were inoculated with Streptomyces sp. MC1, and bioavailable chromium decreased up to 73%.Zea mays seedlings were planted in soil samples contaminated with chromium. Plantlets accumulated chromium mainly as Cr(III), and biomass decreased up to 88%. Streptomyces sp. MC1 was inoculated in soil samples contaminated with 200 mg kg⁻¹ of Cr(VI) and Z.mays seedlings were planted.Streptomyces sp. MC1 caused Z.mays biomass increase (57%), chromium accumulation and bioavailable chromium decreased up to 46% and 96%, respectively.This work constitutes the first contribution of cooperative action between actinobacteria and Z.mays in the bioremediation of Cr(VI) contaminated soil. The large removal capacity of bioavailable chromium by Streptomyces sp. MC1 and Z.mays infers that they could be successfully applied together in bioremediation of soils contaminated with Cr(VI).     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation

Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development.     

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.     

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.     

Effect of FSH on E2/GPR30-mediated mouse oocyte maturation in vitro

Mammalian oocyte restores meiosis can be stimulated by follicle-stimulating hormone (FSH) under normal physiological conditions. G-protein coupled receptor 30 (GPR30), an non-classical estrogen membrane receptor, has been widely reported in teleost oocyte maturation. However, it remains unknown whether GPR30 involves the role of FSH in mammalian cumulus expansion and oocyte maturation. Here, we used mouse cumulus-oocyte complexes (COCs) as a model to investigate how FSH affects the in vitro maturation of mouse oocytes mediated by 17β-estradiol (E₂)/GPR30 signaling. Our study reveals that FSH starts regulating mouse cumulus expansion precisely at 8 h in in vitro culture. ELISA measurement of E₂ levels in culture medium revealed that FSH activated aromatase to promote E₂ production in vitro in cultured mouse COCs. Moreover, the results of real-time quantitative PCR indicated that FSH-induced in vitro maturation of mouse oocytes was regulated by the estrogen-signaling pathway mediated by GPR30; FSH treatment markedly increased the mRNA expression of HAS2, PTGS2, and GREM1 in COCs. Exploration of the underlying mechanism suggested that E₂ produced by mouse COCs regulated the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2) through GPR30 and thereby promoted mouse cumulus-cell expansion and oocyte maturation. In conclusion, our study reveals that FSH induced estrogen production in mouse COCs through aromatase, and that aromatase/GPR30/ERK1/2 signaling is involved in FSH-induced cumulus expansion.     

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me₂SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV⁻ PI⁻), early apoptotic (AV⁺ PI⁻), dead non-apoptotic (AV⁻ PI⁺) and necrotic (AV⁺ PI⁺). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.     

Steroid hormones promote bovine oocyte growth and connection with granulosa cells

Many approaches have been investigated for growing oocytes in vitro in mammals. To support oocyte growth in vitro, the culture systems must meet certain conditions for maintaining connections between oocytes and surrounding granulosa cells. The aims of this study were to determine the effects of combinations of 17β-estradiol (E₂) and androstenedione (A₄) on in vitro growth of bovine oocytes and to determine the number of connections between the oocyte and granulosa cells. Oocyte–granulosa cell complexes (OGCs) collected from early antral follicles (0.4−0.7 mm in diameter) were cultured for 14 days in a medium with different concentrations of E₂ and A₄, either alone or in combinations. We then assessed the number of transzonal projections (TZPs), which extend from granulosa cells through the zona pellucida to the oolemma. During in vitro growth culture, OGC structures were maintained in the medium with steroid hormones. The mean diameter of oocytes grown in the medium with both E₂ and A₄ was increased from 95.8 μm to around 120 μm, larger than oocytes grown without steroid hormones (109.9 μm) and similar in size to in vivo fully grown oocytes (119.4 μm) from 4- to 6-mm antral follicles. In subsequent in vitro maturation culture (22 hours), 30% (12 of 40) and 34% (14 of 41) of oocytes grown with E₂ or A₄ alone, respectively, matured to metaphase II; meanwhile, oocytes grown with a combination of E₂ and A₄ matured to metaphase II at a high rate (58%, 23 of 40). Growing oocytes isolated from early antral follicles had many uniformly distributed TZPs throughout the zona pellucida. After 14 days of culture, there was a significant decrease in the number of TZPs in oocytes grown without steroid hormones, whereas the number of TZPs was maintained in oocytes grown with steroid hormones. In particular, oocytes grown with E₂ alone or with a combination of E₂ and A₄ had numbers of TZPs similar to oocytes before growth culture. In conclusion, a combination of E₂ and A₄ maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes.