Influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus-oocyte complexes during in vitro maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A(1) (with a dense cumulus), A(2) (with a translucent cumulus), B(1) (with the corona radiata), B(2) (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A(1) COCs (p?相似文献   

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Hampered cumulus expansion of porcine cumulus‐oocyte complexes by excessive presence of alpha2‐macroglobulin is likely mediated via inhibition of zinc‐dependent metalloproteases

In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus‐oocyte complexes (COCs) due to excessive alpha₂‐macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc‐dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc‐dependent metalloproteases, the effect of tissue inhibitor of metalloproteases‐3 (TIMP‐3) on cumulus expansion during IVM with and without A2M was investigated. To immuno‐neutralize A2M, serum was pre‐incubated with A2M antibodies. Impaired cumulus expansion because of TIMP‐3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP‐3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP‐3 share a common target, a zinc‐dependent metalloprotease. Future research is directed toward the identification of the protease involved.     

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

The involvement of N‐glycosylation of zona glycoproteins during meiotic maturation in sperm–zona pellucida interactions of porcine denuded oocytes

The present study was conducted to delineate whether N‐glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N‐glycosylation played a role in sperm–ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N‐glycosylation inhibitor, for various intervals during IVM on sperm–ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N‐glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm–ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP‐bound sperm. However, the inhibition of N‐glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N‐glycosylation was involved in the initial stage of fertilization. We conclude that 24–44 h of N‐glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Role of Cumulus Cells on In Vitro Maturation of Canine Oocytes

The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.     

Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap‐frozen cumulus cells, oocytes and day‐7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin‐treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin‐treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over‐maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary

The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2–4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.     

Effect of permeable cryoprotectant‐free vitrification on DNA fragmentation of equine oocyte–cumulus cells

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post‐mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus–oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO₂) before (control group) or after a permeable cryoprotectant‐free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at ?80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant‐free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.     

Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose‐dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6‐AN) and the physiological (NADPH) inhibitors of PPP induced a dose‐dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.     

Formation of an Antral Follicle–like Structure of Bovine Cumulus–Oocyte Complexes Embedded Individually or in Groups in Collagen Gels

Culture techniques of antral follicle–like structure (AFLS) derived from cumulus–oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4–5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4–14 using the individual culture method were significantly higher (p < 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p < 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.     

Effect of the Removal of Cumulus Cells on the Nuclear Maturation, Fertilization and Development of Porcine Oocytes

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.     

Role of the hyaluronan receptor CD44 during porcine oocyte maturation

Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.     

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.     

血清和促性腺激素对山羊卵母细胞核体外成熟的影响

试验研究了清和促性腺激素对山羊卵丘扩展和卵母细胞核成熟的,卵母细胞与卵丘扩展的关系以及卵丘扩展与卵母细胞核成熟的关系。结果表明：（1）培养24h，添加PMSG组卵母细胞核成熟率显著高于不加激素组（P＜0．05），而培养到27h，2者成熟率之间差异变得不显著，说明添加PMSG卵母细胞核的最终成熟率没有明显影响，但加速了卵母细胞核的成熟进程；（2）M199＋BSA培养27h，卵母细胞核成熟率显著高于培养24h，而M199＋FCS培养27h与培养24h的成熟差异不显著，说明添加BSA时，卵母细胞核体外成熟速度比添加FCS的慢；（3）卵丘扩展良好与扩展不好的卵母细胞核成熟率以及第1极体形态无统计学差异，说明山羊卵母细胞的核成熟可能不依赖于卵丘扩展；（4）山羊的卵丘扩展产不依赖于卵母细胞；（5）将带壁颗粒细胞与不带壁颗粒细胞的COC分开培养发现，2者卵母细胞核成熟度无明显差异，带有壁颗粒细胞的COC卵丘扩展情况明显优于一般COC。