Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.     

Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P less than 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P less than 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P less than 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P less than 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture.

Bovine oocytes cultured for 12-20 h in TC-199 were incubated for 24 h in fertilization medium, Brackett and Oliphant medium with bovine serum albumin (10 mg ml-1), caffeine (5 mmoll-1) and heparin (10 micrograms ml-1), with or without frozen-thawed spermatozoa. High penetration rates (93-96%) and significantly (P < 0.001) higher maturation rates were obtained in oocytes incubated with (93-100%) than without (62-72%) spermatozoa. However, when oocytes at the germinal vesicle stage were cultured for 44 h fertilization medium, maturation of oocytes to metaphase II was reduced. However, all oocytes that were first cultured for 20 h and further for 24 h with spermatozoa were penetrated and 40% of the penetrated oocytes reached metaphase II. All of the remaining oocytes that did not mature arrested at the stages of condensed germinal vesicle (39%) or prometaphase I (22%). These results indicate that oocytes at metaphase I at and after sperm penetration are stimulated by sperm penetration to complete maturation.     

Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine

A large number of oocytes are contained in the mammalian ovary. A very small number of these oocytes grow to the final size, mature, and are ovulated. In the ovary there are more early antral follicles than late antral or preovulatory follicles, offering a large pool of oocytes for IVM and IVF if appropriate culture conditions could be devised. In the present study, early antral follicles containing oocytes 90 to 99 microm in diameter were isolated from bovine ovaries. Cumulus-oocyte complexes (COC) with pieces of parietal granulosa (COCG) were then dissected from the follicles. The COCGs were embedded in collagen gels and cultured in Medium 199 with 10% fetal calf serum (FCS) for 8 d. In Experiment 1, the effect of hypoxanthine and FSH on the growth of bovine oocytes was examined. When hypoxanthine (2 and 4 mM) and FSH (10 ng/ml) were added to the culture medium, the number of granulosa cell-enclosed oocytes increased significantly (P < 0.05). All of the oocytes surrounded by granulosa cells showed a normal morphology and were at the germinal vesicle stage, while 75 to 94% of the denuded oocytes were degenerated and had resumed meiosis. The mean diameter of the oocytes showing normal morphology was significantly higher than that measured before culture (P < 0.05). In Experiment 2, the maturational competence of in vitro-grown bovine oocytes was examined. Oocytes which were 90 to 99 microm in diameter before culture did not have meiotic competence. After being in a growth culture of 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 7 or 11 d, granulosa cell-enclosed oocytes were recovered from the COCGs. No significant difference (P < 0.05) in the diameters of the oocytes was observed between 7 and 11 d of culture (7 d: 107.5 +/- 6.1 microm, n = 30; 11 d: 108.0 +/- 5.3 microm, n = 35). After a subsequent 24 h in a maturation free of hypoxanthine and FSH medium, only 17% of the oocytes cultured for 7 d underwent germinal vesicle breakdown. On the other hand, 89% of the oocytes cultured for 11 d underwent germinal vesicle breakdown, and 11% of the oocytes emitted the first polar body and reached metaphase II. These results demonstrate for the first time that bovine oocytes harvested from early antral follicles can grow, and acquire meiotic competence in vitro.     

Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes

Immature red deer (Cervus elaphus ) oocytes (n = 1208) were collected from 1 to 4 - mm diameter follicles on ovaries and then cultured for 16, 20, 24 or 28 h (Groups I to IV) in TCM 199 supplemented with 10% FCS, 1 x 10(6) granulosa cells/ml and 1 mug/ml estradiol at 39 degrees C under 5% CO(2) in air. Gonadotropins (10 mug/ml, FSH and LH) were added to the culture medium at the start of culture (0 h) or after 6 h. Approximately one-third of the oocytes were examined for maturation, and the remainder were fertilized in vitro with frozen-thawed semen collected from a stag by electroejaculation. In vitro fertilized oocytes (n = 309) from four of the maturation treatment (Groups II and III in both gonadotropin treatments) were cultured for 7 d and examined for cleavage. Oocytes cultured for 16 h (Group I) had lower (P < 0.001) maturation rates (4.7%) than those in the longer culture durations (Groups II to IV: 68.9%). Culture for 20 (Group II) and 24 h (Group III) resulted in higher (P <0.001) fertilization rates than culture for 16 (Group I) and 28 h (Group IV) (18.3, 20.5, 7.1, 7.8%, respectively). The time of gonadotropin addition did not affect maturation or fertilization rates, but its addition at 6 h increased (P < 0.05) the percentage of oocytes cleaving (5.7 vs 12.5%). Oocytes cultured for 20 h (Group II) and with the delayed addition of gonadotropins cleaved most readily (18.2%). No embryos developed beyond eight-cell stage.     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.     

Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine

Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 microM BLI (B) or 6.25 microM BLI+12.5 microM ROS (BR) in TCM-199 for 24 h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199+0.5 microg/ml FSH, 50 microg/ml LH and 10% FCS for another 24 h, during which oocytes were fixed every 3 h. In Experiment 2, oocytes were submitted to 24h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21 h in the culture media DMEM+0.5 microg/ml FSH, 50 microg/ml LH, 5% FCS and 50 ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2x10(6) sperm cells/ml) were co-cultured for 18 h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 degrees C and 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0 h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P<0.05). After 3 h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P>0.05) than C (100%, P<0.05). At 12 h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups (approximately 80%, P>0.05). After 21 (77-89%) and 24 h (85-95%), all groups had similar metaphase II (MII) rates of development (P>0.05). In Experiment 2, cleavage (79-84%, P>0.05) and Day 7 blastocyst rates (26-36%, P>0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P<0.05). The other groups were similar to C (35-38%, P>0.05). Hatching rates were similar (10-15%, P>0.05) as were total cell numbers (141-170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates.     

Spontaneous maturation of oocytes isolated from ovaries of immature hypophysectomized rats.

Oocytes were collected from preantral follicles (200-300 mu in diameter) from 30-day-old immature rats 7 days after hypophysectomy. The ova were cultured in vitro for 17 hrs in a chemically defined medium and scored cytologically for meiotic maturation. Of 534 oocytes that were cultured 89% resumed meiosis; however, 98% of these oocytes arrested in either metaphase or anaphase I. In contrast, 82% of the oocytes isolated from preovulatory follicles (approximately 600 mu in diameter) of adult proestrus rats progressed to metaphase II. These results are discussed in terms of functional FSH and LH receptors on the granulosa cells.     

Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro

The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM + 5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM + 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7 +/- 3.1 ng/ml) was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P < 0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.     

Resumption of meiosis in pig oocytes cultured with cumulus and parietal granulosa cells: the effect of protein synthesis inhibition

In denuded and cumulus-enclosed pig oocytes, puromycin at concentrations 5, 10, and 25 micrograms/ml did not lower the rate of germinal vesicle breakdown (GVBD) after 24 h of culture. GVBD was prevented in 50, 75, and 100 micrograms/ml of puromycin. After 40 h of culture, 5 and 10 micrograms puromycin/ml impaired significantly incidence of metaphase II (42 and 30%), respectively. Concentrations of 25 and 50 micrograms puromycin/ml absolutely prevented the first polar body (I PB) expulsion. The results indicated that GVBD in pig oocytes is far less sensitive to puromycin than I PB expulsion. Culture of cumulus-enclosed pig oocytes isolated with a piece of membrana granulosa (C + P oocytes) did not allow GVBD after 24 and 32 h in control medium. After 24 h of culture, GVBD occurred in 43 and 56% of C + P oocytes in the medium supplemented with 17 and 25 micrograms puromycin/ml. GV was broken down in 80 and 68% of C + P oocytes cultured in 17 and 25 micrograms puromycin/ml for 32 h. It is concluded that inhibition of protein synthesis by puromycin released pig oocytes from the block exerted by granulosa cells.     

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.     

Effects of cumulus cells, different cryoprotectants, various maturation stages and preincubation before insemination on developmental capacity of frozen-thawed bovine oocytes

To improve freezability of bovine follicular oocytes, it is necessary to minimize injury to the oocytes caused by freezing and the toxicity of cryoprotectants. The maturing ability of frozen-thawed follicular oocytes with or without cumulus complexes was tested. The proportion of frozen-thawed follicular oocytes reaching the metaphasc II (M II) stage after in vitro maturation of 24 h was significantly (P < 0.05) higher in cumulus oocyte complexes (COCs; 44%) than in denuded oocytes (30%). Oocytes were cultured for 0, 6, 12, 18 or 24 h then frozen-thawed with 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO), and cultured for 24, 18, 12, 6 or 0 h respectively. In PROH, 24:0 (67%) showed significantly (P < 0.05) higher maturation rate than 0:24 (38%), 6:18 (41%). In DMSO, 18:6 (72%) and 24:0 (61%) showed significantly (P < 0.05) higher maturation rate than 0:24 (30%), 6:18 (33%) and 12:12 (44%). In case of 18:6, DMSO (72%) showed significant (P < 0.05) higher maturation rate than PROH (52%), however in case of 0:24, 6:18, 12:12 and 24:0, there was no significant (P < 0.05) difference in the maturation rate between PROH and DMSO. The proportion of embryos developed to > or = 2 cell, > or = 8 cell, morula and blastocyst in 18:6 DMSO (35, 10, 3 and 0%) and 24:0 PROH (38, 12, 5 and 0%) was significantly (P < 0.05) lower than that of fresh oocytes (67, 38, 31 and 16%). There was no significant (P < 0.05) difference in the rate of embryos that developed to > or = 2 cells, > or = 8 cells, morulae and blastocysts between PROH and DMSO. When the frozen oocytes were grouped as rewarming culture (21:2 PROH) and control (24:0 PROH), there was no significant (P < 0.05) difference in the rate of embryos that developed to > or = 2 cells, > or = 8 cells, morulae and blastocysts between 24:0 PROH (42, 24, 11 and 1%) and 21:2 PROH (51, 29, 16 and 4%) but 21:2 PROH showed slightly higher developmental capacity than 24:0 PROH. Transferable blastocysts (4%) were obtained in 21:2 PROH when the frozen-thawed follicular oocytcs were fertilized and cultured for 8 to 9 d.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)