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Collection of Bovine Cumulus-oocyte-complexes (COCs) from Slaughterhouse Ovaries in Bangladesh  [PDF]
M.G. Mostafizur Rahman,P.C. Goswami,M.A.M. Yahia Khndoker,K.M.A. Tareq
Pakistan Journal of Biological Sciences , 2003,
Abstract: Follicles were collected from three categories of ovary obtain from local slaughterhouse. Type-I, having functional corpus luteum (CL); type-II, CL is in almost regressed condition and type-III without CL. The average number of follicles per ovary was 4.37, 5.28 and 6.48, respectively. Significantly higher (p<0.01) number of follicles was obtained from type-III ovaries. The cumulus-oocyte-complexes (COCs) collected from each follicle further classified into 4 grades. The average number of grade-A COCs was 1.71, 2.85 and 3.57 for type-I, type-II and type-III, respectively. Significantly higher (p<0.01) number of grade-A and B COCs was obtained from type-III ovaries. The number of grade C COCs was not varied significantly (p<0.01) among the type. Grade-D COCs was in significantly (p<0.01) higher number in type-III ovaries as an exception of the usual expectation. Considering the diameter of the follicles, no significant (p<0.01) variation was found in the number of follicles measuring between <2 mm and >6 mm diameter in three types of ovary. Grade-A and grade-B COCs collected from 2-6 mm diameter of follicles are usually used for IVM, IVF and IVC experiment. Significantly (p<0.01) higher number of follicles of 2-6 mm diameter was found in type-III ovaries. On the other hand, irrespective of the types of ovaries, grade-A and B of COCs were found to be significantly higher (p<0.01) in follicles of 2-6 mm diameter.
Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro
Michele D Calder, Anita N Caveney, Lawrence C Smith, Andrew J Watson
Reproductive Biology and Endocrinology , 2003, DOI: 10.1186/1477-7827-1-14
Abstract: Generally, cumulus-oocyte complexes (COCs) are collected from 2–8 mm antral follicles from unstimulated bovine ovaries collected from an abattoir for in vitro fertilization procedures. These follicles are several days from reaching pre-ovulatory size and may not have received sufficient exposure to hormones and growth factors in vivo to have the resulting accumulation of maternal mRNAs to develop well in vitro. Recent evidence has shown that maturation condition (oocytes matured in vivo or in vitro) has a significant influence on the numbers of embryos developing to the blastocyst stage [1]. This suggests improvements in maturation media and protocols still could be made that would improve oocyte competence and developmental rate.Although at least 80% of bovine oocytes collected from antral follicles undergo spontaneous nuclear maturation in culture [2], gonadotropins are often added to maturation media to induce cytoplasmic maturation, cumulus expansion and to improve embryonic development. Follicle stimulating hormone (FSH) induces expansion of mouse cumulus oocyte complexes in vitro [3] and improves bovine fertilization and cleavage rate [4]. Luteinizing hormone (LH) has beneficial effects on bovine oocyte maturation [5]. In addition, it has been reported that serum is required for hormonally induced cumulus expansion of COCs, although the percentage may be as low as 0.01–5% [3]. In most cases, supraphysiological hormone concentrations are added to in vitro maturation (IVM) media and it is not clear if these high concentrations are strictly required. Porcine FSH is usually added to IVM media at 0.5–1 μg/ml [2], but up to 10 μg/ml has been used, while reported bovine pre-ovulatory surge FSH concentrations average about 125 ng/ml [6]. Ovine LH is usually added to IVM media at 5 μg/ml [2], but the bovine pre-ovulatory LH surge averages about 200 ng/ml [6]. Recently, recombinant gonadotropins have become commercially available; these are very pure sources of hormone
In Vitro Maturation of Cumulus-Oocyte Complexes for Efficient Isolation of Oocytes from Outbred Deer Mice  [PDF]
Jung Kyu Choi, Xiaoming He
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0056158
Abstract: Background The outbred (as with humans) deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ~5 oocytes per animal can be obtained so far. Objective The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM) of cumulus-oocyte complexes (COCs). Methods Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII) oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF) and embryo development. Results Less than ~5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5) and superovulation (4.3±1.3) in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. Significance We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research.
Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence  [cached]
S Modina,V Borromeo,AM Luciano,V Lodde
European Journal of Histochemistry , 2007, DOI: 10.4081/1139
Abstract: In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH.We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA).We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrineparacrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.
Meiotic Competence of Porcine Oocytes after Percoll Sedimentation Treatment for Oocyte Selection
Morteza Yavari,Akiko Fujii,Ryohei Shimizu,Aya Ito,Yukine Kaedei,Yasuhiro Morita,Fuminori Tanihara,Takeshige Otoi
Journal of Animal and Veterinary Advances , 2012,
Abstract: The objective of this study was to evaluate the effects of Percoll sedimentation treatment of porcine oocytes before In vitro Maturation (IVM) on the meiotic competence of the oocytes. Cumulus-oocyte Complexes (COCs) with uniform ooplasms and compact cumulus cells obtained from porcine ovaries were placed on the surface of Percoll solutions of various concentrations (5, 10, 15, 20 and 25%) in a petri dish. Only the COCs that settled in the dish within 3 min were transferred into maturation medium. At the end of the IVM culture, the nuclear status of the oocytes was assessed. The proportion of COCs that settled decreased with an increase in the concentration of the Percoll solution. When the COCs were treated with less than 10% Percoll solution, more than half the total number of COCs settled. In contrast, the proportion of COCs that settled was lower (p< 0.01) in the 20 and 25% Percoll solutions (12 and 1%, respectively) than in Percoll solutions with lower concentrations (49-90%). The proportion of oocytes that underwent germinal vesicle breakdown and reached metaphase II after the Percoll treatment and subsequent IVM culture did not differ among the 5, 10 and 15% Percoll solution groups. Moreover, it also did not differ from that of the control oocytes that were not treated with Percoll. In conclusion, the selection of oocytes by Percoll sedimentation treatment does not improve the rates of nuclear maturation of porcine oocytes and decreases the total number of COCs available for IVM culture.
GDF-9对牦牛COCs体外成熟培养及其相关基因mRNA表达的影响
Effect of GDF-9 on in vitro maturation culture and the expression of related genes in yak COCs
 [PDF]

王玉恒,徐业芬,索朗斯珠,强巴央宗,郭敏,程玲华,牛家强,贡嘎,王刚,孔庆辉
- , 2018,
Abstract: 为探明生长分化因子-9(Growth differentiation factor 9,GDF-9)对牦牛卵丘-卵母细胞复合体(Cumulus-oocyte complexes,COCs)体外培养的卵母细胞成熟率及相关基因mRNA表达水平的影响:采取健康牦牛的卵巢,分离COCs进行体外成熟培养;在牦牛COCs成熟培养液中添加终浓度分别为0、200、400、600 ng/mL的GDF-9,统计卵母细胞成熟率;采用Real-time PCR方法检测GDF-9处理的COCs中Smad信号通路下游分子Smad 4以及卵丘扩展相关基因(HAS 2、PTX 3、PTGS 2)的mRNA表达水平。结果表明:添加0、200、400、600 ng/mL GDF-9的COCs成熟率差异不显著(P>0.05)。Smad 4和卵丘扩展相关基因(HAS 2、PTX 3、PTGS 2)的mRNA表达量随着GDF-9浓度升高呈上升趋势,其中600 ng/mL GDF-9处理组的Smad 4和PTX 3 mRNA表达量最高,与其他处理组差异显著(P<0.05); 600 ng/mL GDF-9处理组的HAS 2、PTGS 2 mRNA表达量与400 ng/mL GDF-9处理组差异不显著(P>0.05),与其他实验组差异显著(P<0.05)。研究发现,在牦牛COCs体外培养过程中,添加不同浓度的GDF-9对牦牛卵母细胞成熟率的影响差异不显著;GDF-9显著提高其信号通路下游分子Smad 4基因以及卵丘细胞扩展相关基因(HAS 2、PTX 3、PTGS 2)的mRNA表达,说明600 ng/mL GDF-9对牦牛体外培养过程中卵丘扩展有作用,作用机制可能与激活Smad信号通路有关。
The aim of this study was to investigate the effects of growth differentiation factor 9 (GDF-9)on culture and the related gene mRNA expression of yak cumulus-oocyte complexes (COCs) in vitro. The healthy yak ovaries were collected and COCs were isolated for maturation culture in vitro. GDF-9 with final concentrations of 0, 200, 400, and 600 ng/mL was separately added to the maturation medium for yak COCs culture in vitro, and the oocyte maturation rate was separately counted. The mRNA expression levels of Smad 4 and cumulus cell extension related genes (HAS 2, PTX 3, PTGS 2) in COCs were detected by Real-time PCR. The results showed that there was no significant difference in the maturation rates of COCs treated with 0, 200, 400 and 600 ng/mL GDF-9 (P>0.05). The mRNA expression of Smad 4 gene and extension related genes (HAS 2, PTX 3, PTGS 2) were increased with the increasing of GDF-9 concentration. The mRNA expression of Smad 4 and PTX 3 in group treated with 600 ng/mL GDF-9 was the highest, which was significantly different from other treatments (P<0.05). Compared with group treated with 400 ng/mL GDF-9, the mRNA expression of HAS 2 and PTGS 2 in 600 ng/mL GDF-9 group had no significant difference (P>0.05), which was significantly different from other experimental groups (P<0.05). It was found that the maturation rates of yak COCs treated with GDF-9 showed no significant differences, but GDF-9 significantly increased the expression of Smad 4which was the downstream molecule gene of GDF-9 in the cell signaling pathway. GDF-9 also increased the mRNA expression of cumulus cell extension genes (HAS 2, PTX 3, PTGS 2). These results suggested that GDF-9 might play an important role in the extension of cumulus oocytes during culturing yak COCs in vitro, and the mechanism might be related to the
Ascorbic acid effects on in vitro maturation of mouse oocyte with or without cumulus cell
B Nadri, S Zeinoaldini, H Kohram
African Journal of Biotechnology , 2009,
Abstract: Ascorbic acid has long been associated with fertility. This study was designed to determine the effects of ascorbic acid on in vitro maturation of mouse oocyte with or without cumulus cells. In this study, 508 denuded oocytes (DOs) and 527 cumulus–oocyte complexes (COCs) from mice stimulated with pregnant mare’s serum gonadotrophin (PMSG) were incubated for 24 h in medium containing 0, 80, 250 and 750 μ M/ml of ascorbic acid prior to in vitro maturation. Maturation rate was compared. A significant decrease in the maturation rate was observed only when the DOs and COCs were exposed to 750 μ M/ml of ascorbic acid (P < 0.05). The maturation rate in COCs was significantly higher than DOs in all groups (P < 0.05). These results indicate that exposure ascorbic acid promotes the development of mouse DOs and COCs from germinal vesicle breakdown (GVBD) to metaphase II (MII) and prevents cumulus cell degeneration at certain levels, especially 250 μ M/ml of ascorbic acid (P < 0.05). However, further studies on the potential effects of different concentrations of ascorbic acid on oocyte maturation are needed.
Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method
Ta-Chin Lin, Jui-Mei Yen, Tsung-Cheng Kuo, Kun-Bing Gong, Kung-Hao Hsu, Teng-Tsao Hsu
BMC Biotechnology , 2008, DOI: 10.1186/1472-6750-8-38
Abstract: Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored.After the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 μl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group.Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 μl and 6 μl can be an option.The mammalian ovary at birth contains a large store of follicles of which only a small numb
Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation
Pia Lucidi, Nicola Bernabò, Maura Turriani, Barbara Barboni, Mauro Mattioli
Reproductive Biology and Endocrinology , 2003, DOI: 10.1186/1477-7827-1-45
Abstract: In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s) can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique.Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by inhibiting E2 production (P < 0.01). Nevertheless, only fully competent oocytes were able to suppress estrogens production, while those cultured under unfavourable conditions were unable to exert any inhibitory effect on the functions of cumulus cells (P < 0.01).These results demonstrated that good quality oocytes can be easily selected on the basis of their ability to affect granulosa cell steroidogenesis thus reducing failures in reproductive technologies due to the transfer of fertilized oocytes with a scarce ability to sustain embryo development.Oocyte developmental competence, which involves the ability of a germ cell to produce a normal and viable embryo after fertilization, is a condition that results from both nuclear and cytoplasmic maturation. Under natural conditions, the occurrence of maturation is characterized by a high developmental competence of the cell but when this process is carried out in experimental conditions (in vitro or by
Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries – partial results
L.S. Leal,E. Oba,C.B. Fernandes,C.F. Moya
Italian Journal of Animal Science , 2010, DOI: 10.4081/ijas.2007.s2.804
Abstract: Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36o C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5° C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 μl of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.
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