Cytoplasmic control of nuclear maturation in mouse oocytes

Oocytes with germinal vesicles were cut into anucleate and nucleate fragments. At the time of germinal vesicle breakdown (GVBD) in nucleate fragments (after 2–3 h of culture) sister anucleate fragments were fused with the help of inactivated Sendai virus with interphase blastomeres from 2-cell embryos. The hybrid cells were examined after h and 20 h. The anucleate fragments induced chromosome condensation in the nuclei of interphase blastomeres immediately after fusion. On this basis it may be concluded that GVBD and nuclear maturation in mouse oocytes is induced by a cytoplasmic factor which is produced or unmasked independently of the nucleus.     

Nuclear maturation in pig and rabbit oocytes after interspecific fusion

Porcine ovarian oocytes were fused with either homologous (porcine) or heterologous (rabbit) oocytes, both at different stages of maturation. The maturation-promoting factor (MPF) present in maturing porcine oocytes or ovulated rabbit oocytes induced rapid chromosome condensation of the oocytes with intact germinal vesicles (GVs). In the case of activation of ovulated rabbit oocyte, germinal vesicle breakdown (GVBD) of porcine oocytes was incomplete or did not occur. In the giant cells consisting of two immature porcine oocytes, meiotic maturation proceeded in the same manner as in unfused oocytes. However, in cells derived from fusion of immature porcine and rabbit oocytes, two metaphase groups of chromosomes were observed 6 h after fusion. It may be concluded that GVBD is governed after fusion by the cytoplasm originating from the oocytes of more advanced stages of maturation or from those which mature faster.     

Parthenogenetic activation of porcine oocytes after nuclear transfer

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.     

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.     

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1^nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.     

Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence,and subsequent fusion and activation rates after nuclear transfer

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.     

Involvement of thiol-disulfide groups in the sensitivity of fully grown mouse oocytes to calcium-free medium

The lethality caused by calcium-free medium (CFM) to fully grown mouse oocytes significantly decreases if a disulfide reducing agent (dithiothreitol, reduced glutathione, or L-cysteine) is added to the medium. In this condition, most of the surviving oocytes do not spontaneously resume meiosis. We also show that the sulfhydryl content of fully grown oocytes, estimated by monobromobimane labeling, rapidly decreases during culture in CFM. The hypothesis is discussed that lethality of oocytes cultured in CFM may be a consequence of an alteration of thiol-disulfide balance.     

Inhibition of LH-induced and spontaneous meiotic maturation in mouse oocytes by N alpha-tosyl-L-lysine chloromethylketone

The effect of N alpha-tosyl-L-lysine chloromethylketone (TLCK), an inhibitor of trypsin-type proteases, on luteinizing hormone (LH)-induced and spontaneous meiotic maturation and follicular production of cAMP in mice was determined. When follicle-enclosed mouse oocytes were incubated with LH (1 micron/ml), they underwent the breakdown of the germinal vesicle (GVBD). TLCK (0.02-0.5 mM) inhibited LH-induced GVBD in folliculated oocytes. The concentration (0.5 mM) of TLCK that inhibited LH-induced GVBD did not significantly suppress LH-induced cAMP production by follicle cells. The effect of TLCK on spontaneous maturation in cumulus cell-enclosed and denuded oocytes was also determined. TLCK strongly inhibited spontaneous maturation in denuded oocytes only if it was added to the incubation medium for 1-3 h before oocytes were liberated from the follicular tissue. The inhibition of oocyte maturation by TLCK was significantly greater in cumulus cell-enclosed oocytes than in denuded oocytes, either with or without preincubation with TLCK. These results suggest that trypsin-type protease in oocytes participates in the process of meiotic maturation in mouse oocytes.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro

The survival and developmental capacity of cumulus cell-enclosed oocytes frozen (1) at the germinal vesicle (GV) stage, after maturation in vitro with (2) and without (3) FSH, and (4) after gonadotrophin-stimulated ovulation were assessed. Survival, defined as the number of morphologically normal oocytes, after freeze-thaw at the GV stage (69%), was lower than for oocytes frozen after ovulation (84%), and after maturation in vitro with FSH (88%) and without FSH (81%). Treatment with DMSO without freezing had no effect on survival when compared with untreated controls except in immature GV-stage oocytes for which there was a slight reduction. After insemination in vitro, 9% of frozen-thawed GV-stage oocytes cleaved to two equal blastomeres, but none developed to blastocysts. Of oocytes matured in vitro before freezing, 17% cleaved to the 2-cell stage and 18% of these developed to blastocysts. When oocytes were matured in vitro in the presence of FSH, however, the percentage cleaving to the 2-cell stage after freeze-thaw was improved to 55%, and 77% of 2-cell stage embryos developed to blastocysts. When ovulated cumulus cell-enclosed oocytes were frozen, 88% cleaved and 67% of the cleaved embryos developed to blastocysts. When 158 two-cell embryos resulting from oocytes matured in vitro with FSH were transferred to the oviducts of pseudopregnant foster mothers, 41 genetically marked live young were produced (26%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Maturation-inhibiting activity in growing mouse oocytes

Growing mouse oocytes incompetent to resume meiosis were fused with fully grown immature mouse oocytes and cultured for 20-24 h. In giant cells that developed, two intact germinal vesicles remained well conserved in all cases. We propose that the cytoplasm of growing oocytes possesses a maturation-inhibiting activity which is able to arrest, after fusion, nuclear maturation in fully grown oocytes competent to mature spontaneously.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.     

Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium

The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P<0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (>/=10muM 6-AN and >/=10nM DPI) inhibited resumption of meiosis (P<0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P<0.05). More mature oocytes were obtained in the presence of pyruvate+glucose (P<0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P<0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content.     

Timing of nuclear maturation and postovulatory aging in oocytes of in vitro-grown mouse follicles with or without oil overlay

Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0-18 h) and at 3-h intervals during early postovulatory aging (18-27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16-21 h after maturation induction.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.