抑制UCHL1对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响

试验旨在研究抑制泛素羧基末端水解酶-1（UCHL1）对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响。利用DAPI染色、Hoechst染色及SDS-PAGE等方法检测猪卵母细胞体外成熟率、透明带泛素化水平及多精入卵等情况。结果表明,添加不同浓度UCHL1抑制剂（10、20、25、30 μmol/L,二甲基亚砜（DMSO）及对照组）体外培养猪卵母细胞46 h后,对照组成熟率为86.22%,而30 μmol/L UCHL1抑制剂组的成熟率为15.30%,且各处理组间成熟率差异显著（P<0.05）;经Western blotting检测,各处理组的透明带在约61、80、106 ku处均发生不同程度的泛素标记,通过灰度值分析差异显著（P<0.05）;进行体外受精试验后,发现对照组透明带精子黏附数最多,精子入卵数较少,添加30 μmol/L UCHL1抑制剂组的透明带精子黏附数最少,几乎没有精子入卵。结果显示,UCHL1抑制剂对猪卵母细胞的体外成熟有一定影响,随着UCHL1抑制剂浓度的增加,透明带发生泛素化的程度逐渐降低,且UCHL1可调节透明带精子黏附及多精入卵。     

Role of acidification elicited by sialylation and sulfation of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions

The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO(3) treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.     

Inhibition of PSMD4 alters ZP1 ubiquitination state and sperm–oocyte‐binding ability in pigs

The aim of this study was to determine how the duration of culture affects the ubiquitination of zona pellucida (ZP) proteins (ZP1, ZP2 and ZP3) during porcine oocyte maturation in vitro. We analysed the changes in ZP protein ubiquitination under three conditions: (i) during oocyte maturation from stage GV to MII; (ii) in oocytes cultured for different periods of time; and (iii) in oocytes treated with an antibody against PSMD4. Our results show that ZP1 and ZP2 are ubiquitinated at the GV stage, while ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage, and band intensities for these proteins were significantly different between the GV and MII stages (p < .05). We also found that ubiquitination occurs in ZP1, ZP2 and ZP3 after cultured for 46, 52, 58 and 64 hr, and that the level of ubiquitinated ZP1 was significantly different in oocytes that were cultured for different time periods. Finally, treatment with an antibody against PSMD4 resulted in a significant decrease in ZP1 ubiquitination (p < .05), without affecting ZP2 or ZP3. The number of attached sperms per oocyte was also significantly different between control and anti‐PSMD4‐treated groups. Thus, we concluded that ZP1 and ZP2 are ubiquitinated at the GV stage, and ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage. As the duration of culture increases, the ubiquitination levels of ZP proteins decrease. We also found that PSMD4 improves ZP1 ubiquitination during in vitro culture of porcine oocytes and effectively inhibits sperm–oocyte binding.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

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.     

玻璃化冷冻对小鼠卵母细胞透明带的影响

本文旨在通过研究玻璃化冷冻小鼠卵母细胞透明带超微结构、透明带厚度(ZPT)、厚度变量(ZPTV)及双折射分值(ZPB)的影响,分析三者间的相关性,探求玻璃化冷冻液的最佳配方。选用4组应用最广泛的冷冻液配方对小鼠卵母细胞进行处理,与未处理的卵母细胞比较存活率、受精率、卵裂率和囊胚率,选出最适冷冻液配方。以新鲜卵母细胞为对照组,进行冷冻程序但并没有进行实际冷冻的卵母细胞为处理组,进行玻璃化冷冻复苏的卵母细胞为冷冻组,扫描电镜观察各组的透明带超微结构变化;检测3组细胞的ZPT和ZPB,并对ZPT和ZPB、ZPTV和ZPB之间相关性进行分析。结果发现HM+7.5%(DMSO+EG),HM+15%(DMSO+EG)+0.5 mol/L Su冷冻液组对卵母细胞发育潜力影响较小。玻璃化冷冻对ZPT(6.05±0.10μm vs 5.77±0.60μm)和ZPB(0.30±0.38 vs 1.22±0.21)有显著影响(P<0.05),且ZPT与ZPB呈负相关(r=-0.299)(P<0.05)。扫描电镜发现玻璃化冷冻造成卵母细胞透明带呈熔融状,表面凹凸不平,窗孔基本不可见,甚至出现透明带部分脱落的情况;冷冻组透明带超微结构的正常率较对照组和处理组下降,其中粗ZP(44.9%对92.9%和84.8%)(P<0.01)和光滑ZP(31.0%对7.4%和9.1%)(P<0.01)。结果表明,玻璃化冷冻影响卵母细胞的发育潜能,低温对透明带的超微结构有较大的负面影响。     

Roles of the zona pellucida and functional exposure of the sperm‐egg fusion factor ‘IZUMO’ during in vitro fertilization in pigs

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm‐egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP‐intact and ZP‐free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44?0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti‐IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti‐IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP‐free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.     

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida,Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus–oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP’s pores were evaluated in squares of 6.4‐μm width. In group B, oocytes were fertilized in vitro. After 48 h, non‐cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 ± 0.07 μm, which was smaller than the observed on oocytes of quality 2 (0.83 ± 0.10 μm), quality 3 (1.02 ± 0.22 μm) and quality 4 (1.38 ± 0.59 μm) (p ≤ 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p ≤ 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore’s diameter.     

On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.     

没食子酸对黄牛卵母细胞体外成熟的影响

试验旨在研究没食子酸（gallic acid，GA）对黄牛卵母细胞体外成熟及早期胚胎发育的影响，进一步优化黄牛卵母细胞体外成熟体系。在卵母细胞体外成熟液（M液）中添加不同浓度没食子酸（0、10、30、50、100 μmol/L），成熟22~24 h后，统计卵丘扩展情况及卵母细胞成熟率；同时，对成熟的卵母细胞进行正常体外受精（IVF），统计早期胚胎的分裂率、囊胚率、囊胚卵裂球数及卵裂球细胞凋亡率。根据试验结果，选择最优浓度，使卵母细胞在含该浓度没食子酸的成熟液中成熟24 h后，检测其细胞内的活性氧水平（ROS）和总谷胱甘肽（TGSH）含量。结果显示，M液中添加30 μmol/L没食子酸组卵丘扩展分值和成熟率显著高于对照组（0 μmol/L）（P<0.05），其他处理组与对照组无显著差异（P>0.05）；成熟的卵母细胞体外受精后进行后续胚胎培养，其中10和30 μmol/L组的分裂率均显著高于对照组和100 μmol/L组（P<0.05），50和100 μmol/L组分裂率较对照组也有所提高，但差异不显著（P>0.05）；早期囊胚率统计发现，与对照组相比，30和100 μmol/L能够显著提高囊胚发育率（P<0.05），10和50 μmol/L浓度组则无显著差异（P>0.05）；与对照组相比，30、100 μmol/L没食子酸均能显著提高IVF胚胎的早期囊胚卵裂球数（P<0.05）；但囊胚卵裂球凋亡率与对照组无显著差异（P>0.05）；对卵母细胞内活性氧和总谷胱甘肽含量检测时，发现30 μmol/L没食子酸可显著降低细胞内活性氧水平（P<0.05），且显著提高总谷胱甘肽含量（P<0.05）。综上所述，在黄牛卵母细胞体外成熟液中添加适量的没食子酸能有效降低卵母细胞内活性氧水平，提高总谷胱甘肽含量，进而提高卵母细胞成熟的质量及其后续IVF胚胎发育能力。     

猪卵母细胞在GV期与MⅡ期的冷冻保存效果比较研究

为比较猪卵母细胞在GV期与MⅡ期的冷冻保存效果,试验在这两个成熟阶段对其进行玻璃化冷冻,GV期卵母细胞解冻后培养至成熟,MⅡ期卵母细胞解冻后恢复2 h,然后采用免疫荧光标记、Western blotting和链霉蛋白酶溶解方法分别检测它们的皮质颗粒分布、CD9蛋白表达水平和透明带消化时间上的差异。结果表明,GV期卵母细胞在解冻后2 h的存活率显著低于MⅡ期卵母细胞（P<0.05）,但极体排出率与对照卵母细胞无明显差异（P>0.05）;在冷冻MⅡ期卵母细胞中,皮质颗粒的皮质区分布比例和CD9的蛋白表达水平显著下降（P<0.05）,但冷冻GV期卵母细胞经体外成熟后则无明显变化（P>0.05）;冷冻GV期与MⅡ期卵母细胞均不会影响透明带的消化时间（P>0.05）。由此可见,猪卵母细胞在GV期的冷冻存活率虽然较MⅡ期低,但其体外成熟后极体排出率、皮质颗粒分布和CD9蛋白表达水平均未受到冷冻的影响。     

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.     

活性氧对猪卵母细胞SUMO-1表达及其质量的影响

本研究旨在调查活性氧过氧化氢（hydrogen peroxide,H₂O₂）对猪卵母细胞体外成熟（in vitro maturation,IVM）过程中蛋白质类泛素SUMO-1表达及精-卵结合能力的影响。试验分为0（control）、10、50、75和100 μg/mL H₂O₂处理组。利用Western blotting、流式细胞术、实时荧光定量PCR、Hoechst染色等方法检测猪卵母细胞体外成熟、蛋白质类泛素SUMO-1含量、细胞活力、凋亡基因mRNA表达、透明带（zona pellucid,ZP）溶解度和精子-卵母细胞结合的表达。结果表明,75 μg/mL H₂O₂组与对照组、10和50 μg/mL H₂O₂组比较卵母细胞体外成熟率及细胞活力显著降低;75 μg/mL H₂O₂组与其他H₂O₂组相比ZP溶解时间显著延长,并减少了精子黏附在成熟卵母细胞透明带上的数量（P<0.05）。在77和18 ku处出现SUMO-1蛋白标记,75 μg/mL H₂O₂组与对照组、10和50 μg/mL H₂O₂组比较SUMO-1蛋白含量显著降低（P<0.05）。75 μg/mL H₂O₂与对照组、10和50 μg/mL H₂O₂组比较显著下调了Bcl-2基因,而Caspase-3基因表达与对照组和10 μg/mL H₂O₂组比较显著升高（P<0.05）,50 μg/mL H₂O₂与对照组和10 μg/mL H₂O₂组相比显著上调了Bax基因水平（P<0.05）。综上所述,H₂O₂能调控猪卵母细胞体外成熟过程中类泛素化水平以及精-卵结合能力。     

Ultrastructural Study of the Canine Zona Pellucida Surface During In Vitro Maturation

The aim of the present study was to compare the ultrastructure of the surface of the zona pellucida (ZP) of immature and in vitro matured dog oocytes using scanning electron microscopy (SEM). Bitch oocytes were collected after ovariohysterectomy; the ovaries were sliced and the released cumulus–oocyte complexes (COCs) were washed with phosphate buffered saline (PBS). The selected COCs were randomly allocated into three groups, two groups were processed after in vitro maturation at both 72 and 96 h and a third group was processed immediately at immature state in PBS medium. After that, oocytes were fixed, critical point dried and viewed by using SEM. The diameters of the outer holes of the ZP were measured on a total of 93 oocytes; the results were analyzed with anova . The mean diameters of holes were different between groups (p < 0.05): 0.69 ± 0.12, 1.56 ± 0.19 and 1.42 ± 0.27 μm, for immature and in vitro matured oocytes for 72 and 96 h, respectively. The difference in the hole sizes between immature and in vitro matured canine oocytes indicates that the ZP surface is related to oocyte maturity in canines.     

LDL对玻璃化冷冻解冻培养MⅡ期猪卵母细胞线粒体膜电位和透明带泛素化水平的影响

本试验旨在探究添加低密度脂蛋白（low density lipoprotein,LDL）对玻璃化冷冻解冻后MⅡ期猪卵母细胞发育率、线粒体膜电位及透明带泛素化水平的影响。采用线粒体膜电位特异性探针JC-1检测各处理组线粒体膜电位,并采用SDS-PAGE及Western blotting方法分析不同处理组MⅡ期猪卵母细胞透明带蛋白泛素化水平。结果显示,玻璃化冷冻法处理中,10 mg/mL LDL处理组MⅡ期卵母细胞正常率和成熟率（71.92%和69.86%）显著高于对照组（58.26%和54.55%）（P<0.05）,最接近未冷冻组。10 mg/mL LDL处理组MⅡ期卵母细胞线粒体膜电位显著高于对照组、1和20 mg/mL LDL组（P<0.05）。免疫印迹结果显示,未冷冻组和LDL处理组MⅡ期卵母细胞ZP1、ZP2及ZP3蛋白分别在61、80、106 ku发生不同程度的泛素化标记;10 mg/mL LDL处理组ZPs（ZP1、ZP2、ZP3）蛋白泛素化水平显著高于1和20 mg/mL LDL组（P<0.05）,但显著低于非冷冻组（P<0.05）。结果表明LDL可改善玻璃化冷冻解冻培养后MⅡ期猪卵母细胞成熟率、线粒体膜电位及透明带泛素化水平。     

Effect of LDL on the Mitochondrial Transmembrane Potential and ZP Protein Ubiquitination in Vitrified-warmed MⅡ Oocytes

The purpose of this study was to determine the effect of LDL addition on oocyte developmental rate,mitochondrial membrane potential and ZP protein ubiquitination level in MⅡ oocytes from vitrified-warmed GV oocytes.Mitochondrial membrane potentials were labeled with specific probe JC-1,and ZP protein samples of different treatment groups in MⅡ oocytes were analyzed by SDS-PAGE and Western blotting methods. The results showed that normal rate and in vitro maturation of MⅡ oocytes in the 10 mg/mL LDL group (71.92% and 69.86%) were significantly higher than those in control group (58.26% and 54.55%)(P<0.05),that were nearest to non-vitrified group.The mitochondrial membrane potential of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in control group and 1,20 mg/mL LDL groups (P<0.05).The ubiquitinated ZP1,ZP2 and ZP3 of MⅡ oocytes in non-vitrified group and LDL treated groups were labeled at 61,80 and 106 ku,respectively.The ubiquitinated ZPs (ZP1,ZP2 and ZP3) level of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in 1 and 20 mg/mL LDL groups (P<0.05),while significantly lower than that of non-vitrified group (P<0.05). In conlusion, LDL could improve the maturation rate, mitochondrial membrane potential and ubiquitinated ZPs level of MⅡ oocytes from vitrified-warmed GV oocytes.     

不同收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及发育的影响

试验旨在研究不同卵母细胞收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及体外受精的影响。以黄牛为研究对象,采用两种方法（抽卵法和割卵法）抽取卵泡中的卵母细胞,比较两种方法获取的卵母细胞成熟率,结果发现,抽卵法获得的卵母细胞成熟率显著高于割卵法（P<0.05）。将获取的卵母细胞分为4组：A组（对照组,只添加基础成熟培养液）、B组（基础成熟培养液+200 μmol/L半胱氨酸）、C组（基础成熟培养液+20 μg/mL肝素钠）、D组（基础成熟培养液+200 μmol/L半胱氨酸+20 μg/mL肝素钠）,结果发现,D组的卵母细胞成熟率显著高于A、B、C组（P<0.05）,B、C组间卵母细胞成熟率无显著差异（P>0.05）,但两组卵母细胞成熟率均显著高于A组（P<0.05）;A组卵母细胞卵裂率均显著低于B、C、D组（P<0.05）,B、C、D组间卵母细胞卵裂率无显著差异（P>0.05）;D组囊胚率显著高于其他3组（P<0.05）。结果表明,抽卵法获得卵母细胞效率显著高于割卵法,肝素钠及半胱氨酸对黄牛卵母细胞体外成熟和体外受精都有促进作用,且同时添加两种物质对体外成熟的效果更佳。     

Structural and Functional Events on the Porcine Zona Pellucida During Maturation, Fertilization and Embryonic Development: a Scanning Electron Microscopy Analysis

Porcine oocytes and pre-implantation embryos from the same, as well as from different animals, have an extremely heterogeneous morphology of the zona pellucida (ZP) surface, as shown by scanning electron microscopy. For years, it has been believed that this heterogeneous morphology plays an important role in the sperm-oocyte interaction. The aim of this study was to analyse the zona morphology and sperm-binding patterns on the porcine ZP. Oocytes were divided into four categories: immature, matured in vivo, or matured in vitro over a time period of 24 or 48 h. The zona morphology of early embryos grown in vivo or in vitro was also investigated. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between the different kinds of maturation in terms of these zona types. All oocytes exhibited extremely heterogeneous zona morphologies, with no clear trend. During subsequent in vivo embryo development, the zona surface changes from a porous structure to one with a compact surface, while the morphology of in vitro embryos remained compact at all stages of development. The analysis of the number and distribution patterns of spermatozoa trapped in the ZP revealed extremely variable patterns, regardless of the zona morphology. Differences were only present if sorted or unsorted spermatozoa were used for insemination. Regardless of the number of inseminated spermatozoa after sorting, only a few (1-2) could be detected on the ZP. Whether oocytes were matured in vivo or in vitro was not a relevant factor. Unsorted spermatozoa bound in higher numbers than sorted ones. The number was directly dependent on the number of spermatozoa used for insemination.     

Changes in Zona Pellucida Surface after in vivo and in vitro Maturation of Caprine Oocytes

Contents
The zona pellucida (ZP) surface features of ovulated, inmature and in-vitro -matured goat oocytes were evaluated by scanning electron microscopy. The in vitro maturation (IVM) process of the ZP surface of oocytes from prepubertal and adult goats were also compared. Ovulated oocytes were collected from superovulated adult goats. Immature oocytes were recovered from slaughterhouse ovaries of prepubertal and adult goats. In-vitro -matured oocytes from adult and prepubertal goats were obtained after culture in TCM199 supplemented with 20% oestrous goat serum + 10 μg/ml FSH + 10 μg/ml LH + 1 μg/ml estradiol 17β for 27 h at 38.5°C in 5% CO₂ in air. All oocytes were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. Before IVM, the ZP surface of immature oocytes showed a rough surface with tight holes (Type I ZP). After the maturation process, the ZP surface acquired a lattice-like appearance with the outermost layer characterized by the presence of shallower large holes (Type II ZP) . A higher percentage of oocytes showing the mature type II ZP surface was observed in ovulated than in in-vitro -matured oocytes (82.6 versus 56.7%, respectively, p < 0.05). No significant differences were observed in ZP surface features when the IVM process of oocytes (immature and in-vitro -matured oocytes) from adult and prepubertal females was compared. These results show that the morphology of the ZP surface is related to the oocyte maturity in caprine. The IVM process gives rise to an adequate and similar development of the ZP surface in oocytes from adult and prepubertal goats.     

泛素蛋白酶体途径及其在体外受精中的作用研究进展

泛素蛋白酶体途径（ubiquitin proteasome pathway,UPP）是生物体内最主要的蛋白质降解途径,与多种疾病有关,在生物体内具有十分重要的作用,主要降解错误折叠的、多余的蛋白质。UPP是一个循环途径,其主要作用起始于泛素（ubiquitin,Ub）的激活,是受泛素激活酶（ubiquitin-activating enzyme,E1,酵母菌中是UbE1）、泛素结合酶（ubiquitin-conjugating enzyme,E2,UbE2）、泛素连接酶（ubiquitin-protein ligase,E3,UbE3）和去泛素化酶（deubiquitinating enzymes,DUBs）调控的级联过程,最终使被多聚泛素化（polyubiquitination,polyubiquitination,Poly-Ub）标记的蛋白底物在UPP的蛋白水解核心--26S蛋白酶体内被降解成寡肽,寡肽可以进入新的蛋白循环,Ub则由DUBs去除进入下一个UPP循环。UPP在海鞘类和一些哺乳类体外受精（in vitro fertilization,IVF）过程中具有十分重要的作用,能够参与精子获能和顶体反应（acrosomal reaction,AR）,促进精子顶体胞吐,在精卵结合时降解透明带（zona pellucida,ZP）蛋白,辅助精子穿透卵母细胞ZP,促进精子与卵母细胞ZP的融合,从而促使IVF的成功。通过在IVF液中添加一些UPP相关的抗体或抑制剂能够抑制IVF过程中的多精入卵,提高IVF率。作者综述了UPP的主要组成成分及其对IVF的辅助作用及相关抗体的研究进展,以期为今后研究UPP循环在生物体内的作用机制、与生物体内各种疾病的关系、在IVF过程中的作用机制及抑制多精入卵等提供一定的理论依据。