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.     

Susceptibility of bovine germinal vesicle-stage oocytes from antral follicles to direct effects of heat stress in vitro

Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34(cdc2)/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 microM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3-8 mm) were cultured at 38.5 or 41 degrees C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41 degrees C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41 degrees C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41 degrees C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41 degrees C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.     

Confocal microscopy of germinal vesicle-stage equine oocytes

The objectives were to compare cumulus type with nucleus form in equine cumulus oocyte complexes (COCs), to define the percentage of germinal vesicle (GV)-stage oocytes within a population of mares, and to further define GV nucleus shapes of equine oocytes. Cumulus types were as follows: 1) compact (56/208, 26.9%), 2) slightly expanded (37/208, 17.8%), 3) moderately expanded (27/208, 13.0%), 4) greatly expanded (15/208, 7.2%), or 5) denuded (73/208, 35.1%). One hundred thirty of 208 COCs (62.5%) were GV-stage, 21/208 (10.1%) were condensed chromatin-stage, 8/208 (3.8%) were polar body-stage, 40/208 (19.2%) were negative (nonstaining), and 9/208 (4.3%) were fragmented. Cumulus types were associated with nucleus forms because higher proportions (P < 0.05) of GV-stage oocytes occurred in compact (42/56, 75.0%), slightly expanded (30/37, 81.1%), moderately expanded (16/27, 59.3%), or denuded (40/73, 54.8%) COCs than in greatly expanded (2/15, 13.3%) COCs. In contrast, lower proportions (P<0.05) of condensed chromatin-stage oocytes occurred in compact (3/56, 5.4%), slightly expanded (0/37, 0.0%), moderately expanded (3/27, 11.1%) or denuded (9/73, 12.3%) COCs than in greatly expanded (6/15, 40.0%) COCs, and lower proportions (P < 0.05) of polar body-stage oocytes occurred in compact (0/56, 0.0%) or denuded (2/73, 2.7%) COCs than in greatly expanded (3/15, 20.0%) COCs. Germinal vesicle-stage equine oocytes had 4 distinct shapes, with higher proportions (P<0.05) having large-regular (54/130, 41.5%) than scattered (10/130, 7.7%), small-round (29/130, 22.3%), or large-irregular (37/130, 28.5%) shapes. Lower proportions (P<0.05) of large-regular GVs occurred in compact (11/42, 26.2%) COCs than in slightly expanded (15/30, 50.0%), or moderately expanded (12/16, 75.0%) COCs. Therefore oocytes with the large-regular GV shape are probably more advanced in development.     

Meiotic competence of bovine fetal oocytes following in vitro maturation

The in vitro ability between fetal and cow oocytes to resume meiosis and progression to metaphase-II (M-II) was compared. Cumulus oocyte complexes (COCs) were harvested from 2 to 6 mm follicles from ovaries of 7.5 month to term fetuses and adult cows. Cumulus cells were removed using 3 mg/ml hyaluronidase and repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde, stained with DAPI and evaluated under fluorescent microscopy for nuclear status before in vitro maturation (IVM). COCs from fetal and adult ovaries were also matured in 200 microl droplets of medium 199 supplemented with 10 microg/ml FSH, 10/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM hepes and 10% FBS for 24 h at 39 degrees C and 5% CO(2). Matured oocytes were fixed, stained and evaluated as explained above for nuclear status namely stage of germinal vesicle (GV) development and subsequent meiotic competence. Data were analyzed using chi-square analysis. The majority of fetal oocytes (P<0.05) before IVM were at GV stages GV-I (27.7%), GV-II (37.6%) and GV-V (22.8%) compared to cow oocytes, which were at GV stages IV (28.3%) and V (46.7%). After IVM, fewer fetal oocytes were at earlier stages of GV development and majority (P<0.05) were at GV-V (24.0%), premetaphase (17.4%) and metaphase-I (M-I: 7.2%) stages. However, after IVM, more cow oocytes matured to M-II than did fetal oocytes (93.7% versus 26.9%; P<0.05). In conclusion, fetal oocytes do not mature in vitro as well as cow oocytes. Our findings suggest that the low meiotic competence of fetal oocytes can be attributed to their being at earlier stages of GV development before IVM.     

Effects of temperature gradients on in vitro maturation of bovine oocytes

The effect of temperature gradients on in vitro maturation of bovine oocytes was examined in this study. Six treatment groups were made by combining 3 different maturation periods (0 to 10 h, 10 to 18 h and 18 to 24 h) with 2 different culture temperatures (37.0 degrees C and 38.5 degrees C). The frequency of oocytes matured to the metaphase II stage was apparently gradually increased as the culture temperature was increased from 37.0 degrees C to 38.5 degrees C at 0, 10 and 18 h after the onset of culture (75.2 vs 80.5, 82.3 and 84.3%, respectively), but this difference was not significant. Neither was the minor decrease in the proportion of oocytes reaching metaphase II when the temperature was decreased from 38.5 degrees C to 37.0 degrees C at 10 and at 18 h after the onset of maturation (84.3 vs 82.4 and 78.0%, respectively). However, more oocytes cleaved (79.2%; P = 0.0653) and developed to morulae (43.6%; P = 0.0019) and blastocysts (27.4%; P = 0.1568) when they were in vitro matured at 38.5 degrees C between 0 and 10 h, and then at 37.0 degrees C from 10 to 24 h. Although only the morula group was statistically different, cleavage- (79.2 vs 69.8, 72.5, 74.2, 76.3, 74.3%, respectively) and blastocyst formation (27.4 vs 23.2, 24.6, 25.2, 19.6, 21.9%, respectively) from this group was the highest among the 6 treatments.     

Effects of divalent cations on in vitro maturation of bovine oocytes

Lowering the external concentrations of both Mg+2 and Ca+2 caused failure of meiotic resumption in vitro of bovine, oocyte-cumulus complexes. Lowering of external Ca+2 levels singly had no effect on either meiotic resumption or completion of the first meiotic division. Lowering of external Mg+2 concentrations alone, although having no effect on meiotic resumption in vitro when Ca+2 was present, did interfere with the completion of the first meiotic division. The result was arrest of oocyte maturation between germinal vesicle breakdown and formation of the first metaphase plate.     

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes     

Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro

In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10(-8) M, 10(-7) M or 10(-6) M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P < 0.01) decreased the proportion of oocytes developing to metaphase II in a dose-dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10(-7) M and 10(-6) M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P < 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 x 10(-5) M, 5.0 x 10(-5) M and 7.5 x 10(-5) M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P < 0.01) decreased the proportion of metaphase II oocytes when used at 5.0 x 10(-5) or 7.5 x 10(-5) M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I.     

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.     

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.     

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.     

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

The objectives were to evaluate

1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10⁶ cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10⁶ cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Effect of nicotine on in vitro maturation of bovine oocytes

The putative effect of nicotine on maturation and the chromosomal complement of bovine oocytes were investigated in the present study. Cumulus-enclosed oocytes were incubated in maturation medium with 0, 0.5, 1.0, 2.5, 5.0, and 10.0 mmol concentrations of nicotine. The results indicated that: (1) nicotine affected cumulus cell expansion in a dose-dependent manner and the perivitelline space failed to form when concentrations were equal to or greater than 5.0 mmol; (2) oocytes treated with 0.5 and 1.0 mmol nicotine concentrations resulted in maturation rates (83.3% and 85.9%, respectively) which was similar to the control (86.2%), whereas treatment with 2.5 and 5.0 mmol concentrations significantly decreased maturation rates to 70.2% and 26.7%, respectively; (3) nicotine at or over 2.5 mmol caused extremely irregular meiotic spindles and interrupted microfilament organization; (4) chromosomal analyses of oocytes with PB1 showed that oocytes derived from 0.5 and 1.0 mmol nicotine groups had haploid complements similar to the control (87-90%), but when the concentrations were increased to 2.5 and 5.0 mmol the haploid state was significantly reduced to around 70%; (5) oocytes at GVBD (germinal vesicle breakdown) and metaphase I stages were less affected by nicotine at 5.0 and 10.0 mmol concentrations than GV-stage oocytes; (6) maturation rates of the short-term nicotine-treated oocytes could be improved when subsequently incubated in normal maturation medium. Prolonged culture of nicotine-pretreated oocytes resulted in self-activation and some oocytes formed 1 or 2 pronuclei. In conclusion, nicotine affects bovine oocyte cumulus cell expansion, maturation rate, and chromosomal complement in a dose-dependent and an oocyte-stage-dependent manner.     

Heparin effect on in vitro nuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.     

Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro

Follicular oocytes collected prior to the expected time of the LH surge from PMSG-treated immature rats were incubated cummulus-intact (with or without LH) or cumulus-free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm-related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm-related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.     

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)     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.