Activation of in vitro maturing oocytes of the Spanish newt as affected by cycloheximide

The P. waltlii oocytes matured in vitro are activated as a result of cycloheximide (CH) treatment. The female nucleus formation was completed in all activated oocytes within 8 h after the onset of treatment. Activation was induced by 0.5 micrograms/ml CH and 5 micrograms/ml CH induced activation in all treated oocytes.     

The effect of pregnant mare's serum and chorionic gonadotropic hormone on the capacity of bovine oocytes, matured in vitro, for parthenogenetic activation by cycloheximide]

The effect of chorionic gonadotropic hormone (HG) and pregnant mare serum (PMS) on the capacity of cattle oocytes for spontaneous maturation and parthenogenetic formation of the female pronucleus was studied in situ and in vitro. HG added to the culture medium had no effect, irrespective of the dosage. However, perfusion of cow ovaries with HG at a dose of 2 micrograms/ml for 1 h abolished the block of meiosis I in 80% oocytes. About 25% oocytes reached metaphase II (MII) and telophase II (TII); 30% oocytes acquired the capacity to form female pronucleus upon the action of cycloheximide (25 Mg/ml). Perfusion of cow ovaries with PMS at a dose of 5 MU/ml for 1 h resulted in elimination of the block of meiosis I in 95% oocytes; 26% oocytes reached MII and TII, and about 17% oocytes acquired the capacity to form female pronucleus.     

Effect of cycloheximide on nuclear maturation of pig and mouse oocytes

Culture of mouse oocytes in medium with 1 or 100 micrograms cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 micrograms cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 micrograms/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture. The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.     

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.     

Two sensitivity levels of cattle oocytes to puromycin

Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.     

Active maturation-promoting factor is present in mature mouse oocytes

Cytoplasmic extracts of meiotically mature mouse oocytes were injected into immature Xenopus laevis oocytes, which underwent germinal vesicle breakdown within 2 h. Germinal vesicle breakdown was not inhibited by incubation of the Xenopus oocytes in cycloheximide (20 micrograms/ml). Identically prepared extracts of meiotically immature mouse oocytes, arrested at the germinal vesicle stage by dibutyryl cyclic AMP (100 micrograms/ml), did not induce germinal vesicle breakdown in Xenopus oocytes. The results show that maturation-promoting factor activity appears during the course of oocyte maturation in the mouse.     

Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 micrograms/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3, 4, or 5 hours in 2 mM of 6-DMAP and subsequently for 3 hours in 10 micrograms/ml cycloheximide. The incubation in 6-DAMP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked.     

Activation of pig oocytes using calcium ionophore: effect of protein synthesis inhibitor cycloheximide

In vitro matured pig oocytes were activated using a combined treatment of calcium ionophore A 23187 with cycloheximide. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with cycloheximide (0 or 10 microg/ml) for 6 h. Cycloheximide treatment significantly increased the activation rate of oocytes and the percentage of oocytes that were able to develop after activation. The highest activation rate was observed after treatment with 50 microM ionophore. The highest percentage of developing eggs was observed after combined treatment of ionophore (25 microM) with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.     

Time-dependent effects of cycloheximide and alpha-amanitin on meiotic resumption and progression in bovine follicular oocytes

To identify the stage during maturation at which new protein and RNA are synthesized for meiotic resumption, follicular oocytes were cultured in TCM-199 with the protein synthesis inhibitor cycloheximide or the hnRNA synthesis inhibitor alpha-amanitin. Although the meiotic resumption of cumulus-enclosed oocytes was completely blocked by the addition of 25 microg/ml cycloheximide at 4 h after the onset of culture, 23% of oocytes cultured from 5 h post cultivation in the medium with cycloheximide underwent germinal vesicle breakdown (GVBD). By further delaying the addition of cycloheximide, the proportion of oocytes which underwent GVBD increased. Addition of the inhibitor at 8 h or more post cultivation resulted in GVBD occurring in more than 87% of oocytes, though none of them were able to proceed beyond the metaphase I stage. In contrast, the addition of 50 microg/ml alpha-amanitin from the onset of culture significantly reduced the proportion of GVBD to 75% in cumulus-enclosed oocytes, while no significant reduction in the proportions of GVBD was noted in the case of its addition from 1 h of culture onward. However, denuded oocytes were almost insensitive to any treatments with alpha-amanitin. These results indicate that protein synthesis in the oocytes and RNA synthesis in the cumulus cells soon after the onset of culture are necessary for GVBD and that continuous protein synthesis following GVBD is indispensable for progression of the meiotic division in bovine oocytes.     

Effect of cycloheximide upon maturation of bovine oocytes

Germinal vesicle breakdown (GVBD) of bovine oocytes was completely blocked by cycloheximide added to culture medium at concentrations of 1-20 micrograms/ml. Nevertheless, under such conditions a certain degree of chromatin condensation inside the germinal vesicle was observed. The inhibitory effect was not influenced by the presence or absence of cumulus cells and was fully reversible; but the process of GVBD was then significantly accelerated. The critical period in which the proteins necessary for GVBD are synthesized lasts approximately the first 5 h of culture. When germinal vesicle-arrested oocytes are fused to maturing bovine oocytes containing condensed chromosomes, GVBD of immature oocytes occurs within 3 h, even in the presence of cycloheximide. In the mouse, GVBD cannot be inhibited by protein synthesis inhibitors. When immature mouse oocytes are fused with immature bovine oocytes and the giant cells are then cultured in cycloheximide-supplemented medium, both GVs are observed, or only mouse GVBD occurs in common cytoplasm after 8 h of culture. We conclude that protein synthesis is necessary for GVBD of bovine oocytes. Our results also suggest that maturation-promoting factor (MPF) is not autocatalytically amplified in mammalian oocytes.     

The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation

We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control.     

Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Effects of inhibitors of protein synthesis on the ovulatory process of the perfused rat ovary

The effects of two different protein synthesis inhibitors (cycloheximide and puromycin) on the ovulatory process were examined in vitro using a perfused rat ovary model. Ovaries of PMSG (20 i.u.)-primed rats were perfused for 21 h. Release of cyclic adenosine 3',5'-monophosphate (cAMP) and steroids (progesterone, testosterone, and oestradiol) was measured and the number of ovulations was estimated by counting released oocytes. Unstimulated control ovaries did not ovulate whereas addition of LH (0.1 microgram/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 16.7 +/- 3.5 ovulations per treated ovary. Cycloheximide (5 micrograms/ml) totally inhibited the ovulatory effect of LH + IBMX when present from the beginning of the perfusions and also when added 8 h after LH + IBMX. No inhibition was seen when cycloheximide was added 10 h after LH + IBMX (1-1.5 h before the first ovulation; 15.2 +/- 4.4 ovulations per treated ovary). Puromycin (200 micrograms/ml) completely blocked ovulation when present from the beginning of the perfusions and the inhibition was congruent to 60% (6.5 +/- 2.2 ovulations per treated ovary) when the compound was added 8 h after LH + IBMX. Both inhibitors increased LH + IBMX-stimulated cAMP release substantially, but decreased the release of progesterone, testosterone and oestradiol. These results indicate that de-novo protein synthesis is important late in the ovulatory process for follicular rupture to occur.     

In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors

This study evaluated in vitro maturation of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (5 microgram/ml) [DOSAGE ERROR CORRECTED]and butyrolactone I (12.5M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained, and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II revealed that hormonal treatment 2(incubation for 22 h - 10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h - 10 ng EGF/ml) presented higher repeatability percentages: TCM+ PVA (54.5% - 61/112); TCM+ pFF (65.0% - 63/97);NCSU23 + PVA (54.6% - 65/119), and NCSU23 + pFF (58.1% - 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/mland 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups.