Dynamics of morphofunctional changes in aging bovine ova during prolonged culture in vitro

In the absence of activating stimuli, aging processes are initiated in matured mammalian oocytes, which negatively affect the quality of ova and their capacity for further development. On the model of the prolonged culture of bovine oocytes, the dynamics of a number of morphofunctional changes associated with the postovulatory aging was investigated in the present work. In cumulus-enclosed oocytes, migration of the first polar body relative to metaphase II chromosomes started between 18 and 22 h of maturation. The angle of the body deviation from the metaphase plate rose as the culture time increased to 30 h. By 32 h of culture, a gain in the rate of ova with the abnormal chromosome morphology was observed that continued up to 56 h. Furthermore, after 56 h, signs of spontaneous parthenogenetic activation were revealed in 16% of matured oocytes. During the prolonged culture of oocytes deprived of cumulus cells after 20 h of maturation, an increase in the frequency of chromosomal abnormalities was found only by 44 h. At the same time, the cumulus elimination did not affect the maintenance of the meiosis II blockade in aging ova. Meanwhile, destructive chromosomal changes in oocytes were attended by a gradual rise in the level of apoptotic degeneration and reduction in the proliferative activity of surrounding cumulus cells. The results obtained point to the varying temporal dynamics of distinct morphofunctional changes and to the participation of cumulus cells in modulation of the speed of metaphase chromosome destructive modifications in aging bovine ova.     

Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro

The present experiment was to investigate the effect of gonadotropins (LH and hCG), steroids (estradiol and progesterone) and culture media (TCM 199, Ham-F-12, BMOC-3 and modified KRB) on in vitro maturation of cumulus-enclosed bovine oocytes. Oocytes isolated from follicles of 相似文献   

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.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.     

Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes

The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.     

影响山羊体外受精的因素

以屠宰山羊卵母细胞为材料研究了公羊个体、附睾不同部位精子、成熟培养和受精时卵丘存在与否、卵丘扩展程度及卵龄对山羊体外受精的影响。结果表明 :1)不同公羊精液在受精、卵裂和桑椹 /囊胚率上都有显著差异 ;2 )附睾尾精子和鲜精的受精、卵裂和桑椹 /囊胚率无显著差异 ,但显著高于附睾体和附睾头精子 ;3)成熟培养 2 4和 2 7h卵母细胞的的桑椹胚 /囊胚率显著高于培养 2 1和 30h卵母细胞 ;4 )卵丘扩展 3和 4级卵母细胞受精和桑椹胚 /囊胚率显著高于扩展 0和 1级卵母细胞 ;5 )成熟培养前机械去卵丘严重影响卵母细胞体外受精和桑椹胚 /囊胚率 ;6 )受精前完全去掉卵丘显著影响桑椹胚 /囊胚率     

Lanosterol 14alpha-demethylase expression in the mouse ovary and its participation in cumulus-enclosed oocyte spontaneous meiotic maturation in vitro

The expression of lanosterol 14alpha-demethylase (LDM) in the mouse ovary after gonadotrophin administration was examined and the action of follicle fluid meiosis activating sterol (FF-MAS), derived from lanosterol by the action of LDM, on oocyte spontaneous maturation was also evaluated in cumulus cell enclosed oocytes (CEOs). Expression of LDM was primarily in oocytes in primordial and secondary follicles prior to administration of gonadotrophins, but obvious LDM expression was apparent in ovarian somatic cells 48 h after administration of equine chorionic gonadotrophin (eCG), especially in luteal and cumulus cells 54 h after eCG or 48 h after eCG plus 6 h after human chorionic gonadotrophin (hCG). The LDM expression in oocytes was only slightly elevated in larger growing follicles after eCG treatment. On the contrary, 48 h after hCG treatment, the elevated expression of LDM was only detected in interstitial cells. Therefore, eCG may be the primary gonadotrophin for LDM expression, and furthermore for production of FF-MAS in mouse cumulus cells (which are indispensable for oocyte maturation in vivo). Conversely, inhibitors of LDM, either 40 microM azalanstat or 50 microM RS-21745, significantly inhibited oocyte germinal vesicle breakdown (GVB) after 4h of in vitro culture; GVB rates decreased to 14 or 20%, compared to 90% in spontaneous maturation, respectively. There was no significant increase in GVB in CEOs following specific inhibitor of sterol Delta14-reductase and Delta7-reductase, AY9944-A-7 (5-100 microM), until marked oocytes degeneration appeared (50 microM). The phenomena may be ascribed to slow, passive accumulation of FF-MAS by AY9944-A-7, which cannot be associated with fast spontaneous progression. Furthermore, in spontaneous-matured CEOs, LDM was expressed preferentially in cumulus cells instead of oocytes. Therefore, FF-MAS may have a positive role in the spontaneous maturation of CEOs. In conclusion, there was an eCG-dependent dual LDM expression pattern on both oocytes and somatic cells in growing follicles in vivo, which may increase LDM expression and FF-MAS production in cumulus cells for oocyte maturation. For the first time, the inhibitory effect of LDM inhibitors on spontaneous maturation, together with the strong LDM expression in spontaneous matured CEOs, indicated that FF-MAS produced by cumulus cells might participate in spontaneous maturation of mouse CEOs.     

Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes

Immature oocytes were collected from immature female rats (60-65 g) 40 h after injection with 6 IU pregnant mare's serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5-20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development. The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.     

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.     

Fate of the first polar bodies in mouse oocytes

Both nuclear transfer and intracytoplasmic sperm injection (ICSI) practice necessitates studies on the spatial relationship between the MII spindle and the first polar bodies (FPB). Although recent observations have shown that the FPB position does not predict accurately the location of the meiotic spindle in metaphase II oocytes of monkey, hamster, and human, detailed studies on FPB deviation and its affecting factors are lacking. Since polar bodies can be used for genetic testing and oocyte quality grading, their life span under different conditions should be studied. The timing of formation and degeneration and the position relative to the MII spindle of the FPB and the factors affecting FPB deviation and degeneration during in vivo and in vitro aging of both in vivo and in vitro matured mouse oocytes were investigated in this study. Mice of the Kun-ming breed were used, and the intact and degenerated FPB were identified through microscopic morphology in combination with propidium iodide (PI) exclusion test and the chromosomes visualized by Hoechst staining. Results are summarized as follows: (i) oocytes started FPB extrusion at 8 hr after the onset of in vivo or in vitro maturation, but the number of FPB reached maximum much later in vitro (14 hr of culture) than in vivo (10 hr post hCG). (ii) Some FPB began to degenerate before ovulation and around 70% became degenerated within 6 hr after maximal nuclear maturation both in vivo and in vitro; they disappeared faster during in vivo than in vitro aging but turned from intact to degenerated at a similar tempo. (iii) Some FPB began to deviate from the MII spindle 10 hr after hCG injection or in vitro culture and the distance between FPB and the spindle increased with time during both in vivo and in vitro aging. (iv) FPB deviated more slowly in the in vitro matured oocytes than in in vivo matured. (v) Denudation performed after FPB extrusion markedly enhanced its deviation. (vi) The perivitelline space (PVS) increased with time during maturation and aging in vivo and in vitro and the values of PVS and the percentages of FPB adjacent to the spindle were significantly negatively correlated. (vii) Cytochalasin B and colchicine had no effect on FPB deviation. (viii) None of the more than 3,500 FPBs observed was found to be dividing or have divided into two cells at any time points before or after ovulation or in vitro maturation. Our results were consistent with the possibility that the displacement of the FPB was a time- and PVS-dependent process, indicating that PVS would increase with time and its formation and enlargement would facilitate the lateral displacement of the degenerating FPB.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

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

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

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.     

In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus

Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.     

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of aneuploidy frequencies between in vitro matured and unstimulated cycles oocytes by metaphase comparative genomic hybridization (mCGH)

A common observation after in vitro matured oocyte is that they yield poorer embryo quality compared to their in vivo counterparts. This study was designed to assess chromosomal status with metaphase comparative genomic hybridization after in vitro maturation (IVM) in unstimulated cycles and compare the results with those obtained after in vivo maturation. Patients without any obstetrical or gynecological pathology were admitted into the study. IVM oocytes were collected 36 h post hCG and matured in vitro at 37°C in 5% O₂, 6% CO₂, and 89% air for 36 h. All matured (metaphase II) oocytes were subject to polar body 1 (PB-1) biopsy and vitrified individually. PB-1 samples were transferred into 0.25 cc PCR tubes containing 2.5 μl of PBS. PB-1 samples from 12 IVM patients were studied. Twenty-six out of 63 PB-1 samples (41%) were determined as euploid and 37 samples (59%) were aneuploid, whereas these values were 42% euploid and 58% aneuploid in the control group (in vivo matured oocytes). No statistical differences were found between the IVM and the control groups for euploid–aneuploid samples (P = 0.900). More aneuploidy was observed on chromosomes 11, 13, 15, 21, and 22 after IVM. Results show a non-significant rate of abnormal PB-1 formation after IVM compared to in vivo maturation. More aneuploidy was observed in chromosomes 11, 13, 15, 21, and 22 in the IVM group.     

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.     

Effects of cumulus cells on sperm penetration of bovine oocytes in protein-free medium

The present study was conducted to examine the effects of cumulus cells on sperm capacitation, acrosome reaction and penetration of bovine oocytes in vitro in a protein-free medium. In vitro matured oocyte-cumulus complexes (OCCs) and denuded oocytes were co-incubated with spermatozoa in the medium with or without bovine serum albumin (BSA). Higher fertilization rates were obtained in the OCCs (92 and 89%, respectively) than denuded oocytes (57 and 6%, respectively) in the medium with or without BSA (P<0.01). Higher proportion of the denuded oocytes were fertilized in the medium with BSA (57%) than without BSA (6%; P<0.01). These results suggest that the cumulus cells are more effective for increasing fertilization rate than BSA (P<0.05). Both the percentages of capacitated and acrosome-reacted spermatozoa incubated for 4 h with isolated cumulus cells were not significantly different in the medium without cumulus cells in the presence or absence of BSA. The denuded oocytes were inseminated with isolated cumulus cells taken from OCCs matured with or without hormones, follicle stimulating hormone (FSH) and estradiol-17beta (E(2)), and from immature OCCs in a protein-free medium. Presence of the cumulus cells matured with hormones enhanced sperm penetration of denuded oocytes more effectively (81%) than either of the cells matured without hormones (41%) or the immature cells (26%; P<0.01). The conditioned medium of cumulus cells matured with hormones was not effective for sperm penetration of denuded oocytes (2%), while a high proportion (82%) of the oocytes were fertilized when they were inseminated with isolated cumulus cells (P<0.01). In conclusion, the presence of cumulus cells matured with FSH and E(2) was effective for sperm penetration but not for sperm capacitation or acrosome reaction.