Absence Of Cumulus Cells Research Articles

Oxidative Metabolism of Pyruvate Is Required for Meiotic Maturation of Murine Oocytes In Vivo1

The requirement for oxidative metabolism of pyruvate during oogenesis in vivo was evaluated by inactivating Pdha1, a gene encoding an enzymatic subunit of pyruvate dehydrogenase complex, in murine oocytes at the beginning of the follicular growth phase. Immunohistochemical analysis revealed that Pdha1(-) oocytes have dramatically reduced amounts of pyruvate dehydrogenase enzyme by the secondary follicle stage. Despite this reduction, these oocytes grow to normal size, are ovulated, and can be fertilized. Pdha1(-) oocytes are, however, impaired in their ability to support embryonic development, as demonstrated by the failure of fertilized oocytes to develop beyond the one-cell zygote stage in vivo. Immunocytochemical evaluation showed that almost all (98.4%) ovulated Pdha1(-) oocytes have not completed meiotic maturation and/or have gross abnormalities of the meiotic spindle and chromatin. Meiotic maturation is even more compromised when these oocytes are matured in vitro in the absence of cumulus cells or in the presence of the gap junction inhibitor 18-alpha glycyrrhetinic acid, indicating that cumulus cells can partially compensate for this enzymatic deficiency through a gap junction-mediated mechanism. Ovulated Pdha1(-) oocytes were also shown to have reduced levels of total ATP content and NAD(P)H autofluorescence relative to oocytes without this enzymatic deficiency. These studies demonstrate that oxidative metabolism of pyruvate is essential for proper completion of oogenesis, serving as a vital source of energy during meiotic maturation. At earlier stages of oogenesis this metabolic pathway may not be necessary due to metabolic compensation by the granulosa cells.

69 REMOVAL OF CUMULUS CELLS AND PRE-INCUBATION IN CYTOCHALASIN B IMPROVE SURVIVAL OF IMMATURE CAT OOCYTES DURING VITRIFICATION

Vitrification might be the best option to cryopreserve the immature cat oocyte because our recent studies have demonstrated that COCs are (1) highly sensitive to cryoprotectant (CPA) and require short-term exposure to prevent toxicity, and yet (2) resistant to extreme hyperosmotic conditions when pre-incubated in the cytoskeleton stabilizer cytochalasin B (CB). However, surrounding cumulus cells may inhibit the necessary rapid transport of water and CPA across the oocyte membrane during short-term exposure to vitrification solutions (VS) containing high CPA concentrations. The objective was to examine the influence of cumulus cells on oocyte survival during vitrification with or without pre-incubation in CB. Our metric of focus was the chromatin status after warming and IVM. Grade I immature oocytes (n = 420, 4 replicates) were equally allocated to one of four treatments: T1 (removal of cumulus cells in 0.2% hyaluronidase); T2 (pre-incubation in 7.5 µg mL–1 CB for 20 min); T3 (removal of cumulus cells and pre-incubation in CB (as above)); and T4 (control, no removal of cumulus cells and no pre-incubation in CB). After each treatment, half of the oocytes was washed and immediately cultured for IVM (28 h; denuded oocytes being co-cultured with an equal number of fresh COCs to circumvent the absence of cumulus cells). The other half was exposed to 10% (v/v) ethylene glycol (EG) + 10% (v/v) DMSO for 30 s, followed by exposure to VS (20% EG + 20% DMSO + 0.5 m sucrose) for 20 s at room temperature. Oocytes then were vitrified in <1 µL of VS at the tip of a plastic gutter (2 oocytes/gutter) by direct plunge into liquid nitrogen. After 1 day of storage, vitrified oocytes were warmed in 0.25 m sucrose, extensively washed, and cultured for IVM as described above. All oocytes were then fixed and Hoechst-stained to assess their chromatin status. Oocytes subjected to T1, T2, T3, and T4 without vitrification exhibited no degenerated chromatin (clumped or dispersed) after IVM. After vitrification, the incidence of degenerated chromatin was different (P < 0.05; ANOVA) among treatments, with a lower percentage (P < 0.05) when oocytes were subjected to T3 (15.5 � 3.9%; mean � SD) compared to T1 (26.6 � 3.1%), T2 (48.9 � 3.8%), or T4 (71.1 � 3.8%). The percentage of vitrified oocytes reaching the metaphase II stage (MII) also was different (P < 0.05) among treatments with a higher incidence (P < 0.05) in the T3 group (57.8 � 3.9%) compared to T1 (44.6 � 3.7%), T2 (31.1 � 7.7%), or T4 (15.5 � 3.9%) counterparts. Percentages of non-vitrified oocytes reaching the MII were not different (P > 0.05) among treatments (range, 84.7 to 89.0%) but were higher (P < 0.05) compared to vitrified oocytes. The combination of removal of cumulus cells and pre-incubation in CB prior to vitrification had a cumulative, beneficial influence on cat oocyte survival. Interestingly, removal of cumulus cells imparted a greater benefit on oocyte survival than CB pre-incubation alone.

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl–pyrrolidone and 0.4% trehalose and they were warmed in a 0.3 M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly ( P < 0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P < 0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified–warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.

Relationship between equilibration times and the presence of cumulus cells, and effect of Taxol treatment for vitrification of in vitro matured porcine oocytes

The effects of the presence or absence of cumulus cells and equilibration times (1 and 4 min) with cryoprotectant, and Taxol treatment before vitrification (0.5–5.0 μM) on development of in vitro matured porcine oocytes after vitrification were examined. Ethylene glycol (30%) and sucrose (0.5 M) was used as a vitrification solution (39 °C), and cryotop was used for cryo-container. There was a significant relationship ( F value: 6.077, P < 0.05) in the rate of morphologically normal oocytes after vitrification between the equilibration times and the presence or absence of cumulus cells. The blastocyst rates were not significantly different between Taxol (1.4–5.5%) and non-treated control (8.8%). The results show that the optimal exposure time to achieve survival after vitrification depends on the presence or absence of cumulus cells, and that Taxol has no positive effect on the developmental capacity of vitrified, in vitro matured porcine oocytes.

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system

Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.

010. Oocyte control of granulosa cell development and function

Oocytes orchestrate the rate of follicular development and the patterns of gene expression by granulosa cells (GCs). There are two populations of GCs in large antral follicles: mural granulosa cells (MGCs) that line the ovarian follicle wall, and cumulus cells (CCs) closely associated with the oocyte. Subtraction hybridization was used to find transcripts more highly expressed in CCs than MGCs. Among the genes expressed more highly in CCs was one encoding an amino acid transporter (Slc38a3). Slc38a3 mRNA was not detected in oocytes. Expression of Slc38a3 mRNA was reduced in the CCs after removal of the oocyte and restored by co-culturing CCs with fully grown oocytes (FGOs). Alanine is one of the amino acids transported by SLC38A3. This amino acid is poorly transported across the oocyte plasma membrane, but gains access to the oocyte from the cumulus cells via gap junctional communication. Alanine transport into cumulus cells was promoted by paracrine factors secreted by FGOs, but not by growing oocytes (GOs) from preantral follicles. Thus FGOs promote the transport of alanine into CCs, and this amino acid is then passed on to the oocyte via gap junctions. Transcripts encoding enzymes in the glycolytic pathway were also more highly expressed in CCs than MGCs. FGOs, but not GOs, promote elevated expression of some of these transcripts. Likewise, FGOs promote both glycolysis and oxidative phosphorylation by isolated CCs and MGCs. Oocytes do not effectively utilize glucose as an energy source, and oocytes require the presence of CCs to resume meiosis when glucose is the only energy source present. In contrast, oocytes can resume meiosis in the absence of CCs when pyruvate is the sole energy source. Thus oocytes apparently promote glycolysis by their companion granulosa cells to provide energy for their own development. In addition, this may be one way that oocytes coordinate their development with that of follicular somatic components. Supported by Grants HD23839 and HD44416 from the NICHD.

The effects of oocyte storage and cumulus cell presence on canine zona penetration by domestic dog spermatozoa

Zona penetration assays (ZPAs) have been developed in numerous species to evaluate sperm fertilizing potential. Preservation methods to stockpile oocytes would be beneficial because of the difficulty in obtaining sufficient numbers of fresh oocytes. Using a canine ZPA, the objectives of this study were to evaluate: (1) two methods of storing canine oocytes (salt storage and intrafollicular cooling) and (2) the effects of cumulus cells on oocyte penetration. In experiment 1, oocytes from fresh ovaries were assigned at random to 3 categories: fresh control (FRE), salt storage in solution 1 (1.5 M MgCl 2·6H 2O; SS1) and salt storage in solution 2 (0.5 M (NH 4) 2SO 4, 0.75 M MgCl 2·6H 2O, 0.2 mM ZnCl 2; SS2). Each category was subdivided into two treatments: cumulus cells intact (intact) and cumulus cells removed (denuded), resulting in a total of six treatments with n≥15 oocytes per treatment for each ejaculate. Fresh (FRE) intact oocytes demonstrated greater sperm–oocyte interaction than other treatments, including FRE denuded oocytes (11.7±0.6 versus ≤4.1±0.5 sperm–oocyte and 94.9 versus ≤55.6% penetration; P<0.01). Poor sperm–oocyte interaction was demonstrated with all salt-stored oocytes (≤1.6±0.2 sperm–oocyte and <51% penetration), but was further attenuated in the absence of cumulus cells. In experiment 2, oocytes obtained from fresh (FRESH) or cooled (24 h COOL, 48 h COOL) ovaries were used with cumulus cells intact for a total of three treatments with n≥15 oocytes per treatment for each ejaculate. No significant difference was observed in sperm interaction between oocytes from fresh, 24 and 48 h COOL ovaries (12.3±0.5 to 13.1±0.4 sperm–oocyte and 92.2–97.7% penetration; P>0.01). These results indicate that salt storage may cause damage to canine oocytes, subsequently impairing sperm penetration, whereas short-term intrafollicular cooling does not affect the oocyte’s penetrability. Furthermore, greater sperm interaction in oocytes with an intact cumulus suggests a possible role for cumulus cells in canine gamete interaction.

In Vitro Maturation and Ultrastructural Observation of Cryopreserved Minke Whale (Balaenoptera acutorostrata) Follicular Oocytes

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo ( [formula omitted]) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199+10% FBS+5 μg/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 μg/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P<0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P<0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P<0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.

Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate

Strain differences have proven to be crucial components in mouse in vitro fertilization (IVF) and superovulatory protocols. To maximize the yield of IVF-derived mouse eggs, a series of experiments was conducted using different injection timing intervals for administration of pregnant mare serum gonadotropin (PMSG) and hCG to induce follicular development and ovulation. Strains were chosen that were representative of those commonly used in genetic engineering experimentation. These strains included ICR outbred, C57BL/6 inbred, and B6SJLF1 hybrid (C57BL/6J × SJL/J F1) mice. Females were superovulated using 4 PMSG/hCG/IVF timing regimens (group), with sperm obtained from males of the same strain. Group designations were based on the following PMSG/hCG and hCG/oocyte collection intervals, respectively: Group 1, 55 and 21.5 h; Group 2, 60 and 14.5 h; Group 3, 55 and 14.5 h; Group 4, 48 and 14.5 h. After overnight culture of ova, fertilization rates (development to the 2-cell stage) were assessed. A logistic regression was performed using indicator variables for both strain and group. There was a significant strain influence on ova fertilization rate, based on the coefficients of mouse strain (ICR, β = −1.1067, P = 8E-17 and C57BL/6, β = −0.5172, P = 8E-06). Additionally, group affected the proportion of fertilized ova obtained (coefficient of Group 1, β = −1.3152, P = 0.00 and Group 3, β = 0.9531, P = 3E-12). From the coefficients for the interaction terms, the effect of groups varies across mouse strain. Therefore, the treatment that produces the highest fertilization rate is related to and contingent upon the strain of mouse. In the second study, the Group 3 protocol was used to evaluate fertilization differences between cumulus-intact and cumulus-free oocytes. Again, there was a significant strain influence on ova fertilization rate based on the coefficients of mouse strain (ICR, β = −2.6639, P = 0.00; C57BL/6, β = −2.5114, P = 0.00). However, there was no difference between Cumulus and No Cumulus groups (cumulus coefficient, β = 0.1640, P = 0.59872), indicating that there was no affect of cumulus presence on fertilization rate. In summary, responses to standardized mouse IVF protocols vary significantly. The efficiency of IVF procedures can be optimized between and within specific mouse strains by the timing of superovulatory regimens. However, absence of cumulus cells during the IVF procedure does not adversely affect fertilization rate.

Support for the development of bovine embryos in vitro by secretions of bovine trophoblastic vesicles derived in vitro.

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.

In vitro fertilization in mice: Strain differences in response to superovulation protocols and effect of cumulus cell removal

Strain differences have proven to be crucial components in mouse in vitro fertilization (IVF) and superovulatory protocols. To maximize the yield of IVF-derived mouse eggs, a series of experiments was conducted using different injection timing intervals for administration of pregnant mare serum gonadotropin (PMSG) and hCG to induce follicular development and ovulation. Strains were chosen that were representative of those commonly used in genetic engineering experimentation. These strains included ICR outbred, C57BL/6 inbred, and B6SJLF1 hybrid (C57BL/6J × SJL/J F1) mice. Females were superovulated using 4 PMSG/hCG/IVF timing regimens (group), with sperm obtained from males of the same strain. Group designations were based on the following PMSG/hCG and hCG/oocyte collection intervals, respectively: Group 1, 55 and 21.5 h; Group 2, 60 and 14.5 h; Group 3, 55 and 14.5 h; Group 4, 48 and 14.5 h. After overnight culture of ova, fertilization rates (development to the 2-cell stage) were assessed. A logistic regression was performed using indicator variables for both strain and group. There was a significant strain influence on ova fertilization rate, based on the coefficients of mouse strain (ICR, β = −1.1067, P = 8E-17 and C57BL/6, β = −0.5172, P = 8E-06). Additionally, group affected the proportion of fertilized ova obtained (coefficient of Group 1, β = −1.3152, P = 0.00 and Group 3, β = 0.9531, P = 3E-12). From the coefficients for the interaction terms, the effect of groups varies across mouse strain. Therefore, the treatment that produces the highest fertilization rate is related to and contingent upon the strain of mouse. In the second study, the Group 3 protocol was used to evaluate fertilization differences between cumulus-intact and cumulus-free oocytes. Again, there was a significant strain influence on ova fertilization rate based on the coefficients of mouse strain (ICR, β = −2.6639, P = 0.00; C57BL/6, β = −2.5114, P = 0.00). However, there was no difference between Cumulus and No Cumulus groups (cumulus coefficient, β = 0.1640, P = 0.59872), indicating that there was no affect of cumulus presence on fertilization rate. In summary, responses to standardized mouse IVF protocols vary significantly. The efficiency of IVF procedures can be optimized between and within specific mouse strains by the timing of superovulatory regimens. However, absence of cumulus cells during the IVF procedure does not adversely affect fertilization rate.

Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos.

We reported previously on the use of coculture with cumulus cells in insemination medium for the development of human embryos in vitro. Here we describe a prospective trial to determine if this procedure has a significant beneficial effect. On the day after insemination, zygotes were randomized for culture in either a fresh drop of medium without (- cum) or were left in their insemination drop with (+ cum) cumulus cells. Embryos with the best morphological quality were replaced on the third day of development at the eight-cell stage. The remaining embryos were cultured for a further 3 days and cryopreserved if they reached the fully expanded blastocyst (FEB) stage. Three different culture media were used over the period of this study. In 11 patients, supernumerary embryos were available only for continued culture in +cum and three patients had embryos cultured in only -cum. Thirty-nine other patients had embryos assigned to both +cum and -cum treatments. In the +cum group, 98 blastocysts developed from 216 embryos cultured for 6 days (45%), and this was significantly greater (P < 0.01) than the 48 blastocysts from 156 embryos (31%) developing in the absence of cumulus cells. In basal HTF medium (HTF medium with EDTA and glutamine) and basal XI HTF medium (similar to basal HTF but devoid of glucose and phosphate), culture of embryos with cumulus cells produced significantly more FEBs than in the absence of cumulus cells. There was no significant difference between the two culture treatments when regular HTF medium was used. Preliminary results indicate that pronectin-coated dishes provide a good substratum for cumulus cell attachment and embryo development. The culture of human embryos with their cumulus cells in insemination drops of medium produces a significantly greater proportion of FEBs than when the zygotes are transferred to fresh culture drops devoid of cumulus cells. This is the first report of a significantly higher blastocyst rate with coculture in which a real comparison has been made between two culture treatments which differ only in the presence or absence of homologous cumulus cells in insemination drops.

Effects of cumulus cells and follicle-stimulating hormone during in vitro maturation on parthenogenetic activation of bovine oocytes.

The response to parthenogenetic activation (calcium ionophore A23187) of bovine oocytes after 30 hr of culture in different maturation conditions was evaluated. The activation rates of oocytes in response to 10 mu M A23187 were similar (86% +/- 4 vs. 89% +/- 8) when the oocytes matured with or without cumulus cells were cultured in TCM-199 + 10% fetal calf serum (maturation medium [MM]) alone. However, the activation rates were significantly lower in oocytes matured with than without cumulus cells (48 +/- 10 vs. 79% +/- 16; P < 0.05) when MM was supplemented with 0.5 microgram/ml follicle-stimulating hormone (FSH), 50 micrograms/ml luteinizing hormone (LH), and 1.0 microgram/ml estradiol-1 beta (E2). When oocytes with cumulus cells were cultured in different media, MM along, MM + 0.5 microgram/ml FSH, MM + 50 micrograms/ml LH, MM + 1.0 microgram/ml E2, and MM + all three hormones, the percentages of activated oocytes were 91% +/- 3.52% +/- 14.83% +/- 7, 83% +/- 13, and 54% +/- 7, respectively. Oocytes with or without cumulus cells during culture began to form a pronucleus 5 hr after activation (5% +/- 1 and 7% +/- 1, respectively). Finally, the proteins synthesized by oocytes matured with or without cumulus cells before (3 hr) and after (10 hr) activation was labelled with [35S]methionine for analysis. Before activation, a 47 kD protein complex was produced by cumulus cell-intact oocytes. An active protein synthesis of 60 kD was characteristic for the oocytes with cumulus cells cultured in MM containing 0.5 micrograms/ml FSH. After activation, there was an appearance of proteins at 75 kD and 47 kD in the oocytes with cumulus cells but not in the oocytes without cumulus cells during the maturation period. These results indicate that bovine oocytes matured in vitro have different capacities for parthenogenetic activation depending on the presence or absence of cumulus cells and the presence of FSH in MM during culture.

Chronological aspects of the nuclear maturation of bovine oocytes cultivated in vitro

In this study, the chronological changes in the meiotic progress of in vitro maturation of bovine oocytes was analysed. Oocytes (n = 1044) were obtained from a local abattoir and were classified into two groups according to the presence or absence of cumulus cells. They were incubated in microdrops (5 per drop) in TCM-199 (control), TCM-199 + 10% fetal calf serum (treatment 2) and TCM-199 + 10% estrous cow serum (treatment 3). Oocytes were fixed and stained at the end of 0, 6, 12, 18, 24 and 30 h of the beginning of in vitro culture, were evaluated according to the nuclear stage of maturation. These results demonstrated that the immature oocytes at the time of collection (0 h) were in the germinal vesicle stage (GV), and that the highest maturation rate was at 24 h of culture in all treatments. Serum treatments enhanced the maturation rates obtained (52.1 and 55.7%) compared to control (serum-free) medium (42.7%; P < 0.05) in cumulus-cell-enclosed oocytes. In denuded oocytes, the maturation rates were lower compared to cumulus-cells-enclosed oocytes in all treatments. In conclusion, meiotic progression of bovine oocytes can be influenced by the inclusion of sera in the maturation media and by the presence of the cumulus cells.

Effect of cumulus cells and exposure period to ethanol on in vitro development of mouse diploid parthenogenones.

Cumulus-intact and cumulus-free mouse oocytes were exposed to 7% ethanol for 1, 4 and 7 min, and treated with cytochalasin-B. The activation rate and the proportion of diploid parthenogenones in all groups were not significantly different. After 96 hr in culture, a higher number of blastocysts was obtained when either cumulus-intact or cumulus-free oocytes were exposed for shorter times (1 and 4 min) to ethanol. The presence or absence of cumulus cells at activation had no effect on the percentage of blastocysts. However, at 1 and 4 min ethanol-exposure periods, the parthenogenones derived from cumulus-intact oocytes had a higher number of cells than ones derived from cumulus-free oocytes.

Epidermal growth factor enhances porcine oocyte maturation in vitro in the absence of follicular fluid or hormones

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199+10% FBS+5 μg/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 μg/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P<0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P<0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P<0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.

Effect of the number of granulosa cells added to culture medium for in vitro maturation, fertilization and development of bovine oocytes

To induce both in vitro nuclear and cytoplasmic maturation of bovine follicular oocytes, effects were investigated of the presence or absence of cumulus cells, additions of different sera and different numbers of granulosa cells into the maturation medium on fertilizability and subsequent development to the blastocyst stage. A 2×7×3 factorial design experiment was conducted using 2 levels of cumulus cells (with or without cumulus), 7 levels of granulosa cells concentrations (0.0, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0×10 6/ml) and 3 levels of serum (no serum, fetal calf serum; FCS or estrous cow serum; ECS) for 24 hours. In vitro fertilization rates of cumulus-denuded oocytes were lower than those of cumulus-enclosed oocytes (P<0.05). The fertilization rate of oocytes cultured in the medium supplemented with FCS was higher than that of oocytes cultured in the medium supplemented with ECS or in no serum. Oocytes cultured in medium supllemented with granulosa cells and FCS had higher rates of fertilization (P<0.05) and subsequent development to the blastocyst stage (P<0.05) than oocytes cultured without the addition of granulosa cells. The present results indicate that the presence of cumulus cells surrounding immature oocytes and the addition of granulosa cells (1.0 to 7.5×10 6/ml) and FCS to the culture medium may be required to induce both nuclear and cyto-plasmic maturation of bovine oocytes.

Birth of calves after the transfers of oocytes fertilized by sperm injection

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199+10% FBS+5 μg/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 μg/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P<0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P<0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P<0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.