Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization,perivitelline, and intracytoplasmic sperm injections

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4′,6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.     

Nitric oxide is essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro

This study was conducted to determine whether endothelial-derived nitric oxide synthase (eNOS) affects meiotic maturation of mouse oocytes in vitro. Cumulus-oocyte complexes (COC) were isolated from ovarian follicles of 27-day-old PMSG-primed wildtype (WT), and eNOS-knockout (eNOS-KO) females, and cultured in drops of medium under oil at 37 degrees C for 16-18 hr. Experiment 1 was carried out to determine effects of eNOS deficiency on the ability of COC to mature in vitro. To determine whether acute synthesis of nitric oxide (NO) was required for oocyte maturation, COC collected from WT mice were cultured in medium without (control) or with different doses of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS (exp. 2). To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown (GVBD), COC from WT and eNOS-KO females were observed for 3.5 hr. COC from WT females were also incubated in medium without or with L-NAME (exp. 3 and 4). After the culture period, cumulus cells were removed, and oocytes were counted and classified as metaphase II (M II), metaphase I (M I) or showing atypical (degenerative) morphology. To determine viability and nuclear morphology of oocytes, they were stained with fluorescein diacetate or 4,6-diamidine-2'-phenylindole dihydrochloride, respectively. There were no differences in body weights but ovarian weights were lower in eNOS-KO mice compared with WT mice (P < 0.05). Ovaries from eNOS-KO mice contained fewer COC collected relative to WT mice (P < 0.01). Maturation of COC from eNOS-KO mice or WT oocytes treated with L-NAME resulted in a lower percentage of oocytes at M II stage (P < 0.01 and P < 0.05, respectively) and a higher percentage of oocytes at M I or atypical stages compared with those from WT (P < 0.01 and P < 0.05, respectively). Many oocytes that showed either an arrest in M I stage or abnormal morphology were not viable. Several oocytes in M II stage demonstrated abnormalities in distribution of maternal chromosomes. Our data demonstrate that eNOS-derived NO is a key modulator of oocyte meiotic maturation in vitro. These results support our previous observations in vivo and indicate that eNOS/NO has independent functions in both oocyte maturation and follicular/oocyte development.     

Effects of fetuin on zona pellucida hardening,fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment I, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P<0.05) were formed when FCS was used compared to PVA and 0.25mg/ml of fetuin. In Experiment II, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P<0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P<0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P<0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes.     

Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.     

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.     

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.     

A stereological study of medium antral follicles during the bovine estrous cycle

Stereological methods were applied to bovine cumulus-oocyte complexes (COCs) in order to characterize them quantitatively during the estrous cycle. COCs from medium (4-8mm) antral follicles with a compact and complete cumulus mass, and with an uniform or a non-visible ooplasm were aspirated from ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphological types (C1-C3) of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Quantitative data show that COCs appear heterogeneous for all studied parameters. From metestrus to proestrus the volumes of COCs and oocytes remained constant, the volumes of oocytes and oocyte nuclei were correlated, the thickness of the outer zona pellucida decreased, and the relative numerical frequency of follicular type C3 cells increased. Results suggest that COCs from distinct estrus stages are structurally different, with type C3 follicular cells gradually differentiating from cell types C1 and C2.     

Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis

In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.     

卵母细胞透明带异常及其对助孕结局影响的研究进展

人类透明带(zona pellucida,ZP)是在卵泡发生过程中由卵母细胞和颗粒细胞共同分泌的由ZP1-ZP4四种糖蛋白分子组成的高度有序结构,它与卵母细胞的成熟、受精、胚胎发育及妊娠结局等预后紧密关联。许多生殖中心实验室发现有些患者的卵出现全部或部分的透明带异常,而且不同实验室发现的透明带异常类型各异。研究发现这些透明带异常与卵母细胞受精、胚胎发育及临床结局有一定的相关性。本文综述了关于透明带异常及其对卵母细胞受精、胚胎发育潜能和临床结局的影响的研究进展。     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores.     

Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro

In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10(-8) M, 10(-7) M or 10(-6) M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P < 0.01) decreased the proportion of oocytes developing to metaphase II in a dose-dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10(-7) M and 10(-6) M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P < 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 x 10(-5) M, 5.0 x 10(-5) M and 7.5 x 10(-5) M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P < 0.01) decreased the proportion of metaphase II oocytes when used at 5.0 x 10(-5) or 7.5 x 10(-5) M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I.     

Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes

The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg.     

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12–22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.     

HeLa细胞表达分泌重组eGFP-DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1/DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP-DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120 KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子/早胚上定位提供了可行的办法。     

HeLa细胞表达分泌重组eGFP—DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1／DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP—DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子／早胚上定位提供了可行的办法。     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata)

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.