Effects of Growth Hormone on Nuclear Maturation of Ovine Oocytes and Subsequent Embryo Development

The objective of this study was to determine the effect of the presence of recombinant ovine growth hormone either alone or together with follicle stimulating hormone (FSH) during ovine oocyte in vitro maturation (IVM) on nuclear maturation and subsequent embryo development. Moreover, the effect of growth hormine (GH) on embryo development whether influenced by the presence of foetal bovine serum (FBS) was assessed. The abattoir‐derived oocytes were randomly divided into four treatment groups and cultured in maturation medium supplemented with: (i) 0.05 IU/ml FSH; (ii) 300 ng/ml roGH; (iii) FSH + roGH; and (iv) no FSH and GH (control). The percentages of germinal vesicle‐stage oocytes in GH‐treated group after 8 h of culture was significantly higher than the FSH and FSH + GH groups and lower than control (22.4%, 8.7%, 9.1%, and 32% respectively). The percentage of MII‐stage oocytes was significantly increased in the presence of GH after 16 and 24 h of culture compared to the control (44.7% and 83.1% vs 32.6% and 73.6% respectively). There was no significant synergism between GH and FSH in terms of nuclear maturation. The blastocyst rates in serum‐supplemented groups were enhanced by the presence of FSH and GH compared to the control (35.4% and 31.3 vs 11.4% respectively). Compared with either GH or FSH alone, the subsequent embryo development (blastocyst rate), however, was negatively influenced by co‐presence of both hormones (22.8%). In contrast, the corresponding values were not affected in the absence of serum. In conclusion, GH had positive effect on nuclear maturation of sheep oocytes. Moreover, the pattern of the effect of GH on embryo development was influenced by the presence of FBS during IVM.     

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor‐β (TGF‐β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c‐mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ–GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c‐mos [a regulatory subunit of mitogen‐activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M‐phase‐promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)‐test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation. After oocyte transfer, embryonic development was diagnosed at 15 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested, HTF:BME, SOFaa, and TCM 199. In experiment II, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 (±9.6), 23.4 (±8.4), and 13.5 (±12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 ± 22.4) or equine (25.8 ± 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 ± 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.     

Exposure to L-Ascorbic Acid or α-Tocopherol Facilitates the Development of Porcine Denuded Oocytes from Metaphase I to Metaphase II and Prevents Cumulus Cells from Fragmentation

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

In Vitro Maturation of Dromedary (Camelus dromedarius) Oocytes: Effect of Different Protein Supplementations and Epidermal Growth Factor*

The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4‐well culture plates containing 500 μl of the maturation medium and cultured at 38.5°C in an atmosphere of 5% CO₂ in air for 32–36 h. The basic maturation medium consisted of TCM‐199 supplemented with 0.1 mg/ml L‐glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto‐orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase‐I, anaphase‐I (A‐I), metaphase‐II (M‐II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M‐II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M‐II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

Time Course of In Vitro Maturation of Compact Cumulus Horse Oocytes after Roscovitine‐Induced Meiotic Inhibition: Effects on the Coordination Between Nuclear and Cytoplasmic Maturation

This study was carried out to evaluate the usefulness of a pre‐maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre‐cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM‐ROSC) or in a simple medium (M199‐ROSC). After pre‐maturation, oocytes from both the groups were in part denuded and fixed‐stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre‐cultured in M199‐ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM‐ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199‐ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM‐ROSC group (34/90) compared with M199‐ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre‐culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.     

Meiotic Response of In vitro Matured Canine Oocytes under Different Proteins and Heterologous Hormone Supplementation

The impact of TCM‐199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM‐199 supplemented with 25 mM Hepes/l, with 10% heat‐inactivated oestrous cow serum (ECS), 50 μg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22‐μg/ml pyruvic acid, 1.0‐μg/ml oestradiol (E 8875; Sigma), 0.5‐μg/ml follicle‐stimulating hormone (FSH) (Folltropin‐V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 μg/ml oestradiol, or (3) medium supplemented with 1 μg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 μl droplets (up to 25 oocytes per drop) under mineral oil at 37°C in a 100% humidified atmosphere containing 5% CO₂ in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM‐199 culture medium tested did not promote the final steps of IVM of bitch oocytes.     

Effect of Lycopene on Cytoplasmic Maturation of Porcine Oocytes In Vitro

The aim of the present study was to improve cytoplasmic maturation of porcine oocytes by the addition of lycopene into in vitro maturation (IVM) media. We designed six experimental groups; IVM medium was supplemented with 10 IU/ml FSH, FSH and 10 IU/ml human chorionic gonadotrophin (hCG), or FSH and 7 μm lycopene in the first half of the IVM culture (0–22 h) followed by further culture (22–44 h) with or without hCG. The addition of lycopene into IVM media delayed the interruption of communication between an oocyte and the cumulus cells. Although meiotic competence was similar among the six groups, the glutathione level of matured oocytes was significantly higher in the lycopene‐supplemented group (9.89 pmol per oocyte) than that in other groups (7.25 and 7.81 pmol per oocyte). Fertilization rate was significantly improved in lycopene‐supplemented groups (58.3%) more than that in the group supplemented with FSH only (43.1%), whereas there were no differences in developmental competence among the groups (blastocyst rate: 20.1–29.5%). These results indicate that insufficient cytoplasmic maturation during conventional IVM resulted by disconnection of the gap junction between an oocyte and the cumulus cells in the early phase during IVM culture. We concluded that lycopene induced a prolonged sustainment of gap junctional communication between an oocyte and the cumulus cells during porcine IVM culture, which was an effective cytoplasmic maturation of porcine IVM oocytes.     

Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle‐derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.     

Ca2+ Cascade and Meiotic Resumption of the Caprine Primary Oocyte

In this study, we investigated the fluctuations of concentration of intracytoplasmic free Ca(2+) during in vitro maturation of caprine primary oocytes and its role in meiotic resumption. Oocytes that were extracted from caprine ovaries were cultured and allowed to mature in vitro to determine their developmental stages including germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase of the first meiotic division (MI) and metaphase of the second meiotic division (MII). Intracytoplasmic free Ca(2+) turnovers of caprine oocytes at these different developmental stages were measured using the calcium fluorescent probe Fura-2/AM (C(44)H(47)N(3)O(24)) to investigate the dynamics of cytosolic free Ca(2+) during in vitro maturation of oocytes and the role of Ca(2+) in inducing the initiation of meiotic resumption of oocytes. Moreover, the oocytes were cultured in Ca(2+) culture medium and Ca(2+)-free culture medium to examine the effect of extracellular Ca(2+) on the oocyte maturation. The results indicated that Ca(2+) concentrations at GV, GVBD, MI and MII stages were 78.06, 147.41, 126.97 and 97.73 nmol/l, respectively, and that 86.30% of oocytes remained at the GV stage and no oocyte developed to MII in Ca(2+)-free culture medium, and 1.1% of oocytes stayed at the GV stage and 83.5% of oocytes developed to MII in Ca(2+) culture medium. These results suggest that the occurrence of GVBD and cell cycle progression to MI and MII stages are closely related to Ca(2+), and that extracellular Ca(2+) performs a specific function for the initiation of meiotic resumption in caprine oocytes.     

Meiotic Competence of Canine Oocytes Embedded in Collagen Gel

The present study was conducted to examine the meiotic competence of canine oocytes embedded in collagen gel, and to investigate the effects of timed exposure of the oocytes embedded in collagen gel to gonadotrophins during maturation culture, on their nuclear maturation. Cumulus-oocyte complexes (COCs) were collected from bitches at the anoestrous and dioestrous stages of the reproductive cycle. In the first experiment, half of the COCs were embedded in collagen gels. The COCs with or without collagen-gel embedding were cultured in a TCM-199 medium supplemented with 0.1 IU/ml human menopausal gonadotropin (hMG) and 10 IU/ml human chorionic gonadotropin (hCG) for 72 h. In the second experiment, the COCs embedded in collagen gels were cultured in TCM-199 medium with gonadotrophins (hMG and hCG) for various periods (0, 24, 48 and 72 h) and then cultured in the medium without gonadotrophins until reaching total culture period (72 h). The percentage of the oocytes reaching metaphase I and metaphase II (MI/MII) was significantly higher (p < 0.05) in COCs with collagen-gel embedding than in COCs without collagen-gel embedding. The percentage of oocytes that were arrested at the germinal vesicle stage was significantly lower (p < 0.05) in oocytes cultured with gonadotrophins than in oocytes cultured without gonadotrophins. However, there were no significant differences in the percentages of oocytes that reached each stage of meiosis among the groups, irrespective of the duration of exposure to gonadotrophins. These observations indicate that embedding of COCs by collagen gel enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MII stage.     

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.     

Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes

Growth differentiation factor 9 (GDF‐9) and bone morphogenetic protein 15 (BMP‐15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199‐Hepes medium as (a) Control group; (b) GDF‐9 antibody (Ab); (c) BMP‐15 Ab; (d) recombinant human (rh) GDF‐9; (e) rh BMP‐15 or (f) rh BMP‐15 and GDF‐9. Data were evaluated by ANOVA. The Abs against GDF‐9 or BMP‐15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF‐9 Ab (64.4 ± 2.1%) or BMP‐15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF‐9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP‐15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF‐9 (3.7 ± 0.4%) or rhBMP‐15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF‐9 (14.7 ± 3.1) and BMP‐15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.     

Expression of Perilipin 2 (PLIN2) in Porcine Oocytes During Maturation

Perilipins have been reported to limit the interaction of lipases with neutral lipids within the droplets, thereby regulating neutral lipid accumulation and utilization. This study aimed to identify the location and expression of PLIN1 and PLIN2 in porcine oocytes during maturation. Quantitative real‐time polymerase chain reaction (qRT‐PCR), immunostaining and Western blot methods were used to characterize the expression and distribution patterns of PLIN1 and PLIN2 in porcine oocytes. The results showed that PLIN1 was not detectable in porcine oocytes. PLIN2 and BODIPY 493/503‐detected neutral lipid droplets appeared identical distribution patterns and extensive colocalization in both GV and MII porcine oocytes. PLIN2 protein expression was higher in GV oocytes than that in MII oocytes (p < 0.05), although PLIN2 mRNA expression was similar in both groups. These findings suggested that PLIN2 was a major lipid droplet‐associated protein in porcine oocytes.     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。     

In Vitro Ageing of Porcine Oocytes: Changes in Phosphorylation of the Mitogen‐Activated Protein Kinase (MAPK) and Parthenogenetic Activability

The role of mitogen‐activated protein kinase (MAPK) was investigated during ageing of porcine oocytes following in vitro maturation (IVM). Oocytes exhibiting an extruded first polar body after IVM for 46 h (79.3% metaphase II, M II) were used for the experiments. Nuclear maturation stages were not visibly altered after a further 12 h of ageing. Proportion of M II stages (42.9%) decreased significantly whereas fragmentation and degeneration of oocytes increased after an ageing time of 26 h. In vitro ageing for 12 and 26 h led to a significant reduction of MAPK phosphorylation (i.e. activation) compared to oocytes matured for 46 h. When MAPK was inhibited by U0126 in M II oocytes, 30.9% (12 h) and 39.7% (26 h) of oocytes, respectively, left metaphase II arrest and proceeded to early anaphase II. Pronuclear stages or fragmentation could be observed only sporadically (2.6–3.6%). After parthenogenetic activation of oocytes by ethanol/cycloheximide, cleavage stages were reached with rates of 51.9% (46 h IVM), 42.0% (12 h ageing) and 40.3% (26 h ageing), respectively. Furthermore, a significant higher proportion of long‐term aged oocytes (26 h) showed pronuclear formation (8.6%) and fragmentation (7.9%) compared to non‐aged oocytes (each 1.9%). It is concluded that both MAPK phosphorylation and cleavage rate after parthenogenetic activation decreased before alterations of nuclear stages could be detected during in vitro ageing of M II oocytes. A premature MAPK dephosphorylation of M II oocytes caused early anaphase II stages, but cleaved stages could not be achieved.