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.     

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 maturation conditions on spindle morphology in porcine MII oocytes

Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (microm) and spindle area (microm2) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.     

Bone morphogenetic protein 15 supplementation enhances cumulus expansion,nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.     

组织型纤溶酶原激活剂基因在牛卵丘-卵母复合体体外成熟进程中的表达

为了初步了解组织型纤溶酶原激活剂(tissue-plasminogen activator,tPA)在牛卵丘－卵母复合体体外成熟进程中的潜在作用,试验运用实时定量RT-PCR技术分别检测体外成熟过程中不同时间段(0、8、16、24 h)以及添加不同浓度FSH成熟培养16 h后的牛卵丘细胞和卵母细胞中tPA基因相对表达变化,观察添加不同浓度FSH成熟培养16 h后卵丘细胞膨胀程度差异,并统计卵母细胞第一极体排出情况。结果显示,卵丘－卵母复合体在体外成熟初期,tPA mRNA相对表达水平在体外成熟16和24 h的卵丘细胞和卵母细胞中显著高于0和8 h组;在添加不同浓度FSH (0、0.01、0.1和1 IU/mL)体外成熟16 h的各处理组,卵丘细胞中tPA mRNA相对表达量随FSH添加浓度的升高而升高,卵丘细胞的扩散程度亦随之增加;同时tPA mRNA相对表达量在0.01 IU/mL FSH添加组卵母细胞中显著高于其他各FSH添加组。综合以上研究结果,本研究认为,牛卵丘细胞中tPA基因的表达受FSH正向调节;tPA可能促进了卵丘细胞在体外成熟过程中的扩散;同时,tPA在卵母细胞中的表达是否与其第一极体排出有关,需进一步试验验证。     

Effect of Different Manganese Concentrations during in vitro Maturation of Bovine Oocytes on DNA Integrity of Cumulus Cells and Subsequent Embryo Development

Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH‐GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte‐complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures ‘normal’ intracellular GSH content in COCs and protects DNA integrity of cumulus cells.     

The Phosphodiesterase Inhibitor,Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus–Oocyte Complexes Matured in Vitro

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.     

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   

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

The Importance of Having Zinc During In Vitro Maturation of Cattle Cumulus–Oocyte Complex: Role of Cumulus Cells

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.     

Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus–oocytes complexes derived from small follicles

The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3–6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1–2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus–oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF‐derived oocytes than MF‐derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co‐culture of SF‐derived COCs with MF‐derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase‐II stage. Furthermore, the ooplasmic diameter of SF‐derived COCs during IVM was increased to the similar size of MF‐derived those in the presence of MF‐derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co‐culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF‐derived CCMs. In conclusion, we demonstrate that supplementation with MF‐derived CCMs improves the ooplasmic diameter and meiotic competence of SF‐derived oocytes.     

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.     

不同促性腺激素添加方式对猪卵母细胞生发泡染色质构型和卵母细胞成熟能力的影响

在卵母细胞体外成熟培养过程中,培养基中添加激素与否及其激素添加的先后顺序是影响猪卵母细胞核成熟和质成熟的一个重要因素.本试验将猪卵母细胞分别在FSH→不含激素、FSH→LH、FSH LH不含激素中培养48 h(培养第20～22 h后换液),并于成熟培养的第24 h(未换液)、48 h将卵母细胞进行荧光染色,观察其生发泡内染色质构型及卵母细胞核成熟情况.实验表明:(1)在IVM的前24 h,添加FSH LH组的GVIV期卵母细胞比例低于只添加FSH组,但差异不显著(8.99%比17.19%,P＞0.05);(2)在FSH存在的情况下,IVM的前期和后期添加LH能促进卵母细胞发生GVBD;(3)FSH LH培养24 h后转入不含激素培养基组,卵母细胞的核成熟比率显著高于添加FSH组和先添加FSH培养24 h后转入添加LH组(P＜0.05).     

胰岛素对犬卵母细胞体外成熟的影响

研究不同浓度胰岛素及不同培养时间对犬卵母细胞体外成熟率的影响,为改善犬卵母细胞体外培养体系提供参考,采用切割法收集卵巢表面卵丘—卵母细胞复合体(cumulus oocyte complexes, COCs),在含有0.6%葡萄糖的TCM199中添加不同浓度的胰岛素（0、3、6、9 IU/mL）,38.5 ℃、5% CO₂培养箱内成熟培养,观察卵丘扩散程度,剥离卵丘细胞获得裸卵后,室温下固定15 min;Hoechst 33342染色,压片,荧光显微镜下观察核形态,用SPSS 14.0软件统计试验数据。不同浓度胰岛素培养48 h后,各组卵丘细胞扩散效果都不明显;卵母细胞核成熟期没有达到减数分裂中期（MⅡ）,但是6 IU/mL胰岛素组生发泡破裂期（GVBD）比率（35.88%±14.63%）显著高于对照组（11.25%±9.75%）;6 IU/mL胰岛素组延长培养时间至72和96 h后,MⅠ-MⅡ期卵母细胞成熟率分别为13.33%±1.5%、20.8%±1.9%。以上结果表明,犬体外成熟培养基中添加胰岛素既没有提高犬卵母细胞核成熟到MⅡ期,也没有改善犬卵母细胞卵丘扩散效果。但是,在6 IU/mL浓度下延长培养时间,相对增加了成熟率。     

Cumulus Expansion, in vitro Fertilization and Embryonic Development after in vitro Maturation of Bovine Oocytes in the Presence of Follicle Stimulating or Luteinizing Hormone

Contents: Bovine follicular oocytes were matured and fertilize din vitro. The frequency of penetration and subsequent embryonic development were improved considerably, for oocytes cultured in larger volumes allowing larger oocyte groups as compared to the culture of 2 oocytes within 30 μl drops. The effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH), present during in vitro maturation, were studied in terms of cumulus expansion, oocyte penetration, male pronucleus formation and embryonic development. Cumulus expansion including mucification was induced by both hormones. Scanning electron microgfaphs revealed that storage of LH as a frozen solution over a long time period (10 months), destroyed its ability to stimulate cumulus mucification, whereas uncoupling of the cumulus cell processes still occurred. LH caused an increase in the percentage of penetrated oocytes with incomplete sperm head decondensation. This effect was also lost after long term storage. Teh resulting total penetration frequency as well as the proportion of oocytes with both pronuclei formed was now similar to that observed with oocytes matured with fresh LH or FSH. Embryonic development was not altered by the replacement of FSH by LH during in vitro maturation .     

Effect of Follicle Stimulation Hormone and Luteinizing Hormone on Cumulus Cell Expansion and In Vitro Nuclear Maturation of Canine Oocytes

In general, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in the regulation of cumulus cell expansion and oocyte maturation. We investigated the effects of supplementation of FSH or LH in in vitro maturation (IVM) medium on the incidence of cumulus cell expansion and nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with 10% foetal bovine serum (FBS), 1 mg/ml cysteine, 0.2 mm pyruvic acid and different concentrations of FSH or LH (control, 0.5, 5 or 50 microg/ml) at 38.5 degrees C, 5% CO(2) in air for 72 h. The cumulus cell expansion was measured by microscopic visualization, and nuclear maturation of denuded oocytes was determined by staining with 10 microg/ml Hoechst33342 for 30 min. The cumulus cell expansion in the 5 microg/ml FSH group (397.2 +/- 64.3 microm) was significantly higher than those in the control, 0.5, and 50 microg/ml FSH groups (168.3 +/- 19.1, 286.0 +/- 69.7 and 300.0 +/- 84.3 microm, respectively; p < 0.05). However, there was no difference in cumulus cell expansion among the control, 0.5, 5 and 50 microg/ml LH groups (165.6 +/- 20.2, 160 +/- 26.5, 172 +/- 20.5 and 168 +/- 23.1 microm, respectively; p > 0.05). After 72 h of IVM, the proportion of nuclear development to the MI-MII stage in the 0.5 microg/ml FSH group (15.1%) was higher than those in the control, 0.5 and 50 microg/ml FSH groups (0.9%, 6.5% and 8.0%, respectively; p < 0.05). However, there was no significant difference in nuclear maturation to the MI-MII stage among control, 0.5, 5 and 50 microg/ml LH groups (4.6%, 2.3%, 5.4% and 8.6%, respectively; p > 0.05). This study indicated that a FSH supplement in IVM medium can increase cumulus cell expansion and nuclear maturation, while the nuclear maturation rate remained low. Further studies are required to improve the nuclear development to the MI-MII stages in canine oocytes.