319 OXYGEN CONSUMPTION OF BOVINE CUMULUS CELLS AND OOCYTES CULTURED IN DIFFERENT CULTURE SYSTEMS FOR OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 267 ◽  
Author(s):  
H. Abe ◽  
H. Shiku ◽  
S. Aoyagi ◽  
T. Matsue ◽  
H. Hoshi
Keyword(s):  
Oxygen Consumption ◽  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Measuring System ◽  
Electron Microscopic ◽  
Respiration Activity ◽  
Serum Free ◽  
Consumption Rates

Oxygen consumption is a ubiquitous parameter that can provide valuable information on metabolic mechanisms and on oocyte and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of bovine cumulus cells and oocytes cultured in serum-free and serum-supplemented media for oocyte maturation. Bovine cumulus–oocyte complexes (COCs) were obtained from ovarian follicles 2–6 mm in diameter. COCs were cultured in IVMD101 medium for serum-free culture and HPM199 medium supplemented with 5% calf serum (HPM199+CS) for serum-supplemented culture in a humidified atmosphere of 5% CO2 in air (20% O2) at 38.5°C for 24 h. Oxygen consumption by single COCs was non-invasively quantified by a SECM measuring system (Abe et al. 2004 J. Mamm. Ova Res. 21, 22). After the measurements, COCs were treated with 0.5% pronase to completely remove the cumulus cells. The oxygen consumption of single denuded oocyte was measured by SECM. Some COCs and oocytes were prepared for transmission electron microscopy. Oxygen consumption has been monitored in COCs and oocytes cultured in IVMD101 and HPM199+CS media for oocyte maturation (Table 1). Oxygen consumption rates of the immature COCs and denuded oocytes (immediately upon recovery from ovary: control) were 6.91 and 0.70 (×10−14 mol s−1), respectively. In serum-free culture (IVMD101), an increase in oxygen consumption rate was found in oocytes, whereas the oxygen consumption of COCs decreased during oocyte maturation. On the other hand, the oxygen consumption of COCs and oocytes cultured in serum-supplemented medium (HPM199+CS) were not change compared with that of controls. Electron microscopic study demonstrated that the mitochondria moved from a peripheral location in the ooplasm to an even spatial distribution in the oocytes cultured in IVMD101 medium, whereas many of the mitochondria in oocytes cultured in HPM199+CS were distributed in the peripheral region of the ooplasm after oocyte maturation. These results suggest that the respiration activity of bovine cumulus cells and oocytes changed during oocyte maturation, and the respiration activity and ultrastructural features of oocytes may affect the culture conditions. The SECM procedures may provide valuable information on oocyte quality and culture conditions for oocyte maturation. Table 1. Oxygen consumption rates (F × 10−14 mol s−1) of the bovine COCs and oocytes in oocyte maturation cultures

scholarly journals 129 RESPIRATION ACTIVITY OF BOVINE EMBRYOS CULTURED IN SERUM-FREE AND SERUM-CONTAINING MEDIA

2005 ◽  
Vol 17 (2) ◽  
pp. 215 ◽  
Author(s):  
H. Abe ◽  
H. Shiku ◽  
S. Aoyagi ◽  
T. Matsue ◽  
H. Hoshi
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Consumption Rate ◽  
Embryo Quality ◽  
Consumption Rate ◽  
Developmental Stages ◽  
Culture Conditions ◽  
Bovine Embryos ◽  
Respiration Activity ◽  
Serum Free ◽  
Consumption Rates

Oxygen consumption is a ubiquitous parameter which can provide valuable information about metabolic mechanisms and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by the scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of individual bovine embryos at different developmental stages cultured in serum-free and serum-supplemented media. Bovine oocytes were matured in IVMD101 medium [Research Institute for the Functional Peptides (IFP), Shimojo, Yamagata, Japan] and inseminated in BO-based medium. For serum-free culture, inseminated ooocytes were cultured to the blastocyst stage in IVD101 medium in an atmosphere of a low oxygen condition (5% CO2/5% O2/90% N2) at 38.5°C. For serum-supplemented culture, inseminated oocytes were cultured in HPM199 medium (IFP) supplemented with 5% calf serum (HPM199 + CS) in the presence of bovine cumulus/granulosa cells in a humidified atmosphere of 5% CO2 in air. Oxygen consumption by individual bovine embryos was non-invasively quantified by the SECM measuring system. Some embryos were prepared for transmission electron microscopy. The oxygen consumption rates are presented in the table. Oxygen consumption rates (F) of the single embryos were low from 2-cell to 8-cell stages (0.45–0.52 × 10−14 mol s−1). In serum-free culture, an increase in oxygen consumption rate was found at the morula (1.03 × 10−14 mol s−1) stage, and blastocysts showed an even higher oxygen consumption rate (1.86 × 10−14 mol s−1). On the other hand, the oxygen consumption of morulae and blastocysts produced in serum-supplemented medium was lower than that of embryos cultured in serum-free medium. Electron microscopic study demonstrated that many of the mitochondria of morulae and blastocycts cultured in HPM199 + CS medium were an immature form, indicating a correlation between respiration activity and development of mitochondria. These results suggest that the culture conditions affect the respiration activity of bovine embryos. The SECM procedures may have a wide application for judging embryo quality and culture conditions for embryos. Table 1. Oxygen consumption rates (F × 10−14 mol s−1) of the bovine embryos at various developmental stages

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

22 RESPIRATORY ACTIVITY AND ULTRASTRUCTURAL FEATURES OF BOVINE SOMATIC NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 129
Author(s):  
H. Abe ◽  
K. Aoyagi ◽  
S. Aoyagi ◽  
H. Shiku ◽  
T. Matsue ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Nuclear Transfer ◽  
Respiratory Activity ◽  
Measuring System ◽  
Ionophore A23187 ◽  
Bovine Embryos ◽  
Single Donor ◽  
Close Relationship ◽  
Consumption Rates ◽  
Functional Peptides

We succeeded in determining oxygen consumption of individual bovine embryos non-invasively and quantitatively by scanning electrochemical microscopy (SECM). Recently, we have found that there is a close relationship between high oxygen consumption and developmental ability of bovine IVF embryos. However, the relationship between the respiratory activity and the quality of bovine embryos reconstructed by somatic cell nuclear transfer (SCNT) is still unclear. The aims of this study were: (1) to assess the oxygen consumption of single bovine SCNT and IVF embryos; and (2) to examine the ultrastructural features of SCNT embryos. Bovine oocytes were matured in IVMD101 medium (Research Institute for the Functional Peptides, Yamagata, Japan) and enucleated. The recipient oocytes were activated by treatment with Ca ionophore A23187 and then incubated with IVMD101 containing cycloheximide. Single donor cells (fibroblasts derived from an adult cow) were placed into the perivitelline space of the enucleated oocytes and the two cells were fused by electrofusion. The nuclear transfer embryos were cultured in IVD101 medium without bovine cumulus/granulosa cell co-culture in a humidified atomosphere of 5% CO2/95% air at 38.5�C. Oxygen consumption rates by whole embryos were quantified individually by the SECM measuring system. Some of these embryos were prepared for observation by transmission electron microscopy. Statistical analyses were performed using one-way analysis of variance (ANOVA) and Fisher

216 THE RELATIONSHIP BETWEEN OXYGEN CONSUMPTION RATE AND PREGNANCY RATE OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 225 ◽  
Author(s):  
N. Sakagami ◽  
K. Akiyama ◽  
Y. Nakazawa
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Embryo Quality ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Consumption Rates ◽  
The Relationship

A precise evaluation of embryo quality is important to estimate the suitability of embryo transfer to recipient animal. Recently, an objective evaluation method was reported for bovine embryos, in which the oxygen consumption of embryos can be noninvasively determined by scanning electrochemical microscopy (SECM) (Shiku et al. 2001 Anal. Chem. 73, 3751–3758). Trimarchi et al. (2000 Biol. Reprod. 62, 1866–1874) suggested that the oxygen consumption reflects the cell number and mitochondrial activity of embryos. The objectives of this study were (1) to examine the oxygen consumption of in vivo-derived embryos by SECM, (2) to investigate the relationship between oxygen consumption and morphological estimation of embryos, and (3) to assess the correlation among the oxygen consumption, embryo viability, and pregnancy rates. Fifty-six embryos were collected from Japanese Black cattle, which were superovulated with a total dose of 20 mg porcine FSH (FSH-R; Kawasaki Pharmaceutical Co., Ltd., Tokyo, Japan) followed by AI. The qualities of collected embryos at the stage of compacted morulae (CM), early blastocysts (EB), and blastocysts (BL) on Day 7 after AI were categorized as grade 1 and grade 2, according to the IETS manual (2002). The oxygen consumption rates of embryos were evaluated by SECM, as previously described by Abe et al. (2004 J. Mamm. Ova Res. 21). Embryos were frozen by programmable freezer in Dulbecco&apos;s PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20&percnt; calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30&deg;C water bath for 15 s. After thawing, the embryos were examined for oxygen consumption. Twenty-eight embryos were then cultured in TCM-199 supplemented with 20&percnt; fetal bovine serum and 0.1 mM &beta;-mercaptoethanol for 24 h to assess the viability of embryos by re-expansion of blastocole. The remaining 28 embryos were transferred to recipients. The pregnancy rates were determined by rectal palpation on Day 70. Data were analyzed by ANOVA. The consumption rates of BL embryos on Day 7 were significantly higher (P &lt; 0.05) than those of CM collected on the same day (0.84 vs. 1.29 &times; 10&minus;14 mol s&minus;1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P &lt; 0.05). After freezing&ndash;thawing, the average oxygen consumption rates of embryos were 0.52 &times; 10&minus;14 mol s&minus;1 for CM (n &equals; 9), 0.67 &times; 10&minus;14 mol s&minus;1 for EB (n &equals; 8), and 0.96 &times; 10&minus;14 mol s&minus;1 for BL (n &equals; 11). The CM embryos with rates of &lt; 0.5 &times; 10&minus;14 mol s&minus;1 and the EB and BL embryos with those &lt; 0.6 &times; 10&minus;14 mol s&minus;1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates &lt;0.5 &times; 10&minus;14 mol s&minus;1 for CM (n &equals; 5) and &lt;0.7 &times; 10&minus;14 mol s&minus;1 for EB (n &equals; 9). A high pregnancy rate (67&percnt;) was obtained from embryos with rates &gt;1.0 &times; 10&minus;14 mol s&minus;1 for BL (n &equals; 14). These results suggest that the measurement of oxygen consumption of embryos after embryo freezing and prior to embryo transfer may be useful for estimating embryo quality and suitability of embryo transfer.

339 DIFFERENTIAL MECHANISM OF LEPTIN ACTION IMPROVING NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 277 ◽  
Author(s):  
F. F. Paula-Lopes ◽  
M. Boelhauve ◽  
F. Habermann ◽  
F. Sinowatz ◽  
E. Wolf
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Serum Free Medium ◽  
Free Medium ◽  
Blastocyst Development ◽  
Serum Free ◽  
Nuclear Maturation ◽  
Polar Bodies ◽  
Series Of Experiments

Leptin is a pleiotrophic peptide that has been implicated in the events associated with oocyte maturation and acquisition of developmental competence. Previous studies indicated that leptin supplementation during oocyte maturation has long-term effects, increasing blastocyst development and reducing the proportion of TUNEL-positive cells per blastocyst. Moreover, blastocysts derived from leptin-treated oocytes showed increased BIRC4 and reduced BAX expression (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). The objective of the current study was to determine the mechanism of leptin action during bovine oocyte maturation. In the first series of experiments cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0, 1, or 10 ng/mL leptin or 10% estrous cow serum (ECS) as positive control. Leptin reduced the proportion of cumulus cells undergoing cell death through apoptosis as determined by TUNEL staining (6.8 � 0.4, 1.8 � 0.4, 1.5 � 0.4, and 6.3 � 0.5% for 0, 1, or 10 ng/mL leptin or 10% ECS, respectively; P < 0.0001), but had no effect on the frequency of apoptotic oocytes. Nuclear maturation was also enhanced by leptin. The proportion of oocytes with extruded polar bodies (64.0 � 2.9, 75.7 � 2.9, 73.8 � 2.9, and 63.3 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) and the proportion of DAPI-stained metaphase II oocytes (72.9 � 2.9, 90.5 � 2.9, 85.8 � 2.9, and 82.0 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) were increased by 1 and 10 ng/mL leptin. There was no effect of ECS in any of the parameters examined. A second series of experiments tested whether the maturation-promoting activity of leptin was mediated by cumulus cells. Denuded oocytes (DO) and COCs were matured in serum-free medium containing 0 or 10 ng/mL leptin. The percentage of oocytes with extruded polar bodies (COCs: 59.1 � 3.7% vs. 75.7 � 3.7%, and DO: 45.5 � 3.7% vs. 54.8 � 3.7% for 0 or 10 ng/mL leptin; P < 0.01) was increased by leptin regardless of cumulus cells. Even though leptin did not affect cleavage rate, it increased blastocyst development. The proportion of COCs that developed to the blastocyst stage increased from 22.3 � 4.6% in the control group to 35.2 � 4.1% in the leptin-treated group. On the other hand, DO matured without cumulus cells did not acquire developmental competency, and this was not reversed by leptin supplementation (1.0 � 5.0% vs. 1.0 � 4.7% for 0 or 10 ng/mL leptin; P < 0.05; leptin � oocyte interaction P < 0.05). In conclusion, leptin enhanced oocyte maturation by acting directly in the oocyte and indirectly through cumulus cells. This suggests that leptin modulates the release of cumulus-derived factors secreted in the oocyte through gap junction coupling and in the extracellular environment.

241 STRATEGIES TO IMPROVE GLUTATHIONE CONTENT OF IN VITRO-MATURED BOVINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 200 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Colorimetric Assay ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Experimental Conditions ◽  
Cell Counts

Glutathione is the main non-enzymatic defense against oxidative stress and a critical part of oocyte maturation and normal fertilization. Our aim was to test different strategies to manipulate cellular glutathione (GSH) content of bovine in-vitro-matured (IVM) oocytes and study the development of embryos produced from such oocytes. The reducing agents lipoic acid (LA, intracellular) and dihydrolipoic acid (DHLA, extracellular) were compared to the cell-permeable reduced glutathione (GSH) donor glutathione ethyl ester (OET) for their effect on oocyte GSH content, oocyte maturation, and blastocyst development (OET only). Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured for 24 h in a humidified atmosphere of 6% CO2 at 38.5�C in modified tissue culture medium (mTCM199) supplemented with 2% (LA, DHLA) or 10% (OET) fetal calf serum (FCS; Gibco, Grand Island, NY, USA), 0.1 IU bLH and 0.1 IU bFSH (Sioux Biochemicals, Sioux City, IA, USA). COCs were matured in the presence of either LA (100 µm) or DHLA (100 µm) alone or in combination with L-cystine (CYS; 0.6 mm), CYS alone, or OET at 1, 3, and 5 mm. COCs matured under control and experimental conditions were denuded of cumulus cells (40 IU hyaluronidase) and scored for maturity. GSH content of MII oocytes was determined by colorimetric assay (Northwest Life Science Specialties, LLC, Vancouver, WA, USA). Oocytes matured in OET were inseminated with frozen/thawed bull sperm (2 � 106 mL-1), cultured to the blastocyst stage (COOK bovine medium, COOK Australia, Brisbane, Queensland, Australia), and subjected to differential cell count (propidium iodide/Hoechst). GSH levels (mean � SEM) and developmental data (percentage) are expressed for n = 18–73 oocytes or embryos and were analyzed by ANOVA or chi-square test (significance, P ≤ 0.05). LA alone failed to increase oocyte GSH content over 2% FCS control levels (6.98 � 0.22 pmol/oocyte v. 5.26 � 0.4 pmol/oocyte). DHLA alone significantly increased oocyte GSH content (9.64 � 0.8 pmol/oocyte) compared to both LA and controls (10% FCS; 4.78 � 0.36 pmol/oocyte). CYS alone (10.18 � 0.58 pmol/oocyte) or in combination with LA (10.84 � 0.37 pmol/oocyte) or DHLA (9.75 � 0.66 pmol/oocyte) significantly increased GSH compared to controls. GSH content of MII oocytes matured in 5 mm OET (8.35 � 0.35 pmol/oocyte) was significantly higher compared to control (5.07 � 0.32 pmol/oocyte), 1 mm (4.21 � 0.18 pmol/oocyte), and 3 mm (7.12 � 0.35 pmol/oocyte) OET treatments. Maturation rates of oocytes were significantly reduced in 2% FCS (51.1–72%) compared to 10% FCS (90.5%). OET treatment (1–5 mm) did not significantly alter maturation rate compared to control (75–89.8%). Blastocyst development of IVM oocytes treated with 1 mm OET (22.5%) was significantly lower compared to 3 mm (42.3%) and 5 mm (41.1%) OET but not to control (33.6%). Blastocysts from IVM oocytes treated with 5 mm OET had significantly higher cell counts compared to controls (126 � 6.4 cells v. 100.8 � 5.2 cells). Bovine IVM is a valuable model for testing the efficacy of various strategies to increase oocyte cellular GSH. Both strategies improve oocyte GSH levels, and an increase in blastocyst cell number occurred with GSH donor treatment (5 mm OET).

scholarly journals Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 317-326 ◽  
Author(s):  
Masaki Yokoo ◽  
Paisan Tienthai ◽  
Naoko Kimura ◽  
Koji Niwa ◽  
Eimei Sato ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Binding Ability ◽  
Migration Ability ◽  
And Migration ◽  
Boar Spermatozoa

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

Energy substrates and amino acids provided during in vitro maturation of bovine oocytes alter acquisition of developmental competence

Zygote ◽  
1998 ◽  
Vol 6 (4) ◽  
pp. 285-294 ◽  
Author(s):  
T.A. Rose-Hellekant ◽  
E.A. Libersky-Williamson ◽  
B.D. Bavister
Keyword(s):  
Amino Acids ◽  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Basic Medium ◽  
Blastocyst Development ◽  
Energy Substrates ◽  
Cumulus Expansion

Energy substrates and amino acids were evaluated for supporting acquisition of developmental competence by bovine cumulus-oocyte complexes during in vitro maturation. The basic culture medium (Basic Medium-3) used for in vitro maturation of oocytes was modified to produce six media containing glucose or glutamine with lactate or pyruvate, or glucose + glutamine, or glucose + 11 amino acids; a seventh (control) medium was TCM199. All media contained polyvinyl alcohol, gonadotropins, epidermal growth factor and oestradiol. Following maturation, oocytes were incubated in medium TALP for fertilisation, then cumulus cells were removed and presumptive embryos cultured for 48 h in a chemically defined medium (HECM-6) followed by 120 h in medium TCM199 + bovine calf serum. Six substrate treatments yielded similar first cleavage responses (66-78%) at 72 h post-insemination; however, blastocyst development at 192 h varied significantly. Oocytes matured in medium with glucose + 11 amino acids gave the best blastocyst development: 21% of inseminated oocytes or 25% of 2-cell embryos. Cumulus expansion in HECM-6 required glucose with either glutamine, 11 amino acids or lactate, or glutamine + lactate. We conclude that (1) the type of energy substrate or nutrient supplied during in vitro maturation of oocytes profoundly affects subsequent developmental competence; (2) oocyte maturation in simple medium containing glucose with lactate or 11 amino acids or glutamine, or lactate + glutamine, can support development equally as well as the complex medium, TCM199; and (3) media supporting at least moderate cumulus expansion during oocyte maturation also support subsequent blastocyst development.

Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI)

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 277-282 ◽  
Author(s):  
Yukiko Yamazaki ◽  
Teruhiko Wakayama ◽  
Ryuzo Yanagimachi
Keyword(s):  
Oocyte Maturation ◽  
Intracytoplasmic Sperm Injection ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Serum Free ◽  
Nuclear Maturation ◽  
Normal Term ◽  
Developmental Capacity ◽  
Oocyte Cytoplasm

The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.

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