Morphological profiles of mouse ovarian follicles: extensive accumulation of a strongly negative-charged substance at specific foci in follicular tissue during oocyte maturation

Structural changes in cumulus-oocyte complexes and granulosa cells were induced by administering pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to female mice. Three and 9 h after hCG administration, dissolution of the germinal vesicle (GVBD) and progression to the second metaphase occurred respectively in oocytes. The number of cumulus cells associated with the granulosa cell layer decreased significantly 1 h after hCG administration. Expansion of cumulus cell investment was due to the abundant deposition of intercellular materials in the cumulus-oocyte complexes during oocyte maturation. A strongly negative-charged (colloidal iron-positive) substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Moreover, cells located where the cumulus mass and granulosa cell layer intertwined became enlarged during the resumption of the meiosis of oocytes. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.     

Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo-matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro-matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro-matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro-matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro-matured oocytes were dissolved within 131.7 +/- 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro-matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for beta-D-Gal(1-3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro-matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro-matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro-matured and in vivo-matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional black to polyspermy in pig oocytes.     

Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare?

In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes to analyse the stages of nuclear maturation. The transfer technique did not impair significantly the final maturation of the recipient preovulatory follicle. The indigenous oocytes within the recipient follicles were recognisable by their larger expanded cumulus of yellow colouration due to high hyaluronic acid content; 7/12 of these oocytes were mature (metaphase II). Around half (42/86; 49%) of the oocytes transferred to preovulatory follicles were recovered subsequently. Most of them showed cumulus expansion (41/42, 6 of which were rich in hyaluronic acid), 13 (32%) were mature, 15 (36%) were immature and 13 (32%) were degenerate. When the indigenous oocyte of the recipient mare was mature, 38% of the transferred oocytes were mature, this rate being no different from the in vitro maturation rate of 46%. This study showed that in vivo maturation of immature oocytes by transfer into a preovulatory follicle in a recipient mare is possible. The maturation rate is not different from the in vitro maturation rate. The technique allows the generation of mature oocytes that have an expanded cumulus rich in hyaluronic acid, similar to the situation in preovulatory oocytes. This result has not been obtained in vitro previously.     

Free innervated latissimus dorsi muscle flap for reconstruction of full-thickness abdominal wall defects

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.     

Oocyte maturation and intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI), treatment of severe male infertility allows an accurate evaluation of oocyte maturity at recovery after corona-cell removal. In cycles comprising a GnRH analog desensitization and a stimulation by hMG or FSH, 12% of oocytes aspirated from follicles (> 14 mm), 34 hours post-hCG are still immature, in prophase or metaphase 1. They are able to achieve meiosis in vitro in 66% of the cases and will be fertilized (2 PN) by ICSI in 51% of the cases as the in vivo mature oocytes of the same cohort. Nevertheless, the quality of cytoplasmic maturation and consequently of embryonic viability remains to be assessed as there still are few pregnancies arising from in vitro matured oocytes. ICSI also represents the only way to obtain normal fertilization in some exceptional but observed anomalies of oocyte maturation, particularly when there is a lack of zona reaction leading to repetitive polyspermy in conventional IVF.     

Effect of follicular cells, IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

Protein neosynthesis by porcine oocytes matured in vivo and in vitro

The protein pattern of individual porcine oocytes matured as intact cumulus oocyte complexes either in vivo or in vitro with or without FSH and LH for 43 h were investigated. The synthesis of a 53 kDa polypeptide ceased 21 h after hCG administration whereas a 44 kDa polypeptide were consistently absent in the protein patterns of nearly all of the in vivo maturing oocytes. Further on, a polypeptide with a relative molecular weight of 46000 persisted throughout maturation. A precipitous increase in the synthesis of two other proteins with relative molecular weights of 38000 and 28000, respectively, was observed at 9 and 21 h after hCG injection. In in vitro matured oocytes with or without FSH and LH the synthesis of the 53 kDa band decreased after a culture period of 9h. Further on, the production of the 44 kDa polypeptide ceased only in oocytes incubated in FSH and LH supplemented media after 21 h of culture. On the other hand, the two proteins of Mr 38000 and 28000 appeared only in most of the protein profiles of oocytes cultured with FSH and LH for 43 h. The production of the 46 kDa polypeptide during a 21 h culture period was significantly affected by the presence of additional granulosa cells in connection with the cumulus oocyte complex. Neither the appearance nor the disappearance of the 5 investigated bands was influenced by the presence or absence of the germinal vesicle after 21 h of culture. It is concluded that at least the addition of FSH and LH to the culture medium is necessary for cumulus oocytes complexes to synthesize a protein pattern closely corresponding to that produced by in vivo matured oocytes.     

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.     

Therapeutic drug monitoring: antiarrhythmic drugs

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

Development of calcium signalling mechanisms during maturation of human oocytes

Sperm-induced Ca2+ signals mediate the events of oocyte activation at fertilization. In this study, the development of mechanisms involved in the generation of Ca2+ signals in human oocytes was investigated. The thiol reagent, thimerosal, which induces oscillations of intracellular Ca2+ ([Ca2+]i) similar to those seen during fertilization, was used to mobilize Ca2+ in in-vivo matured, immature and in-vitro matured human oocytes. There was an increase in the sensitivity to thimerosal during maturation of human oocytes, with oocytes from small antral follicles being relatively insensitive, compared with those from luteinized follicles, which displayed a large spike followed by sustained oscillations in [Ca2+]i. These oscillations were inhibited by caffeine which suggests that they were mediated by the inositol trisphosphate receptor Ca2+ release system. When immature oocytes were cultured in vitro they acquired the capacity to undergo a single large spike in [Ca2+]i, however, subsequent sustained oscillations were not observed, indicating that these oocytes failed to develop fully competent Ca2+ signalling mechanisms during culture in vitro. This finding may be a key factor in the poor developmental competence of in-vitro matured human oocytes.     

Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report

OBJECTIVE: To report a normal pregnancy and the delivery of a healthy child after the combination of in vitro maturation of germinal-vesicle stage oocytes and intracytoplasmic sperm injection (ICSI) in a patient. SETTING: Procedures were performed in a tertiary IVF center coupled with an institutional research environment. MAIN OUTCOME MEASURES: Maturation rate of immature oocytes after in vitro maturation and intactness, fertilization, and developmental rates of oocytes after microinjection. RESULTS: Nine of 14 germinal-vesicle stage oocytes matured to the metaphase II stage after 30 hours of in vitro culture (64%). Seven of eight injected and intact oocytes fertilized normally (78%) and five of them cleaved with < 20% fragmentation (71%). Four embryos were transferred and a singleton pregnancy was obtained that ended in the delivery of a healthy child. CONCLUSION: In vitro maturation of immature oocytes together with ICSI can result in normal fertilization, embryo development, pregnancy, and the delivery of healthy child.     

Gonadotropins, serum, and amino acids alter nuclear maturation, cumulus expansion, and oocyte morphology in hamster cumulus-oocyte complexes in vitro

Glucose, lactate, and pyruvate (the substrate triad), gonadotropins, serum, and amino acids were tested on maturation of cumulus-oocyte complexes (COCs) using a simple defined medium, Tyrode's-PVA (T-PVA). In experiment 1, effects of FSH (2 microg/ml) and the substrate triad were tested using a 2 x 2 factorial design. After 12-13 h, nuclear maturation was depressed in the absence of the triad or with FSH (0-14% metaphase II [MII]) compared with the triad alone (92% MII, p < 0.05). Subsequent experiments used as the base medium Tyrode's solution with the triad (TLP-PVA): adding 10% bovine calf serum (BCS) and gonadotropins (10 microg/ml FSH, 10 microg/ml LH, or both) yielded nuclear maturation equivalent to that in medium alone (88-100% post-metaphase I [post-MI] oocytes). Responses with glutamine, or with 11 but not 20 amino acids, were equivalent to the response in BCS with gonadotropins (93-100% post-MI oocytes). Some cumulus expansion occurred in COCs matured with gonadotropins and BCS, or glutamine, or 11 amino acids, but was less extensive than for in vivo-matured COCs. Oocytes matured with gonadotropins and BCS, or glutamine, or 11 amino acids plus gonadotropins, but not medium alone, had normal-appearing first polar bodies. Another cytoplasmic marker, cortical distribution of microfilaments (detected by confocal microscopy), did not differ between in vitro- and in vivo-matured oocytes. We conclude that effects of gonadotropins on hamster nuclear maturation, cumulus expansion, and oocyte morphology are modulated by serum or amino acids; maturation conditions producing normal oocyte and cumulus morphologies are predicted to yield developmentally competent oocytes.     

Ischemic cerebral vascular accident caused by vertebral artery dissection]

The present study examined the time-dependent effects of follicular cells on the fertilizability of oocytes and their subsequent development to blastocysts. The percentages of oocytes reaching the metaphase-II stage of maturation rose from 51.3% after 16 h of culture to 86.2% at 28 h (cumulus-intact oocytes; CIO) and, for the same time points, from 65.4% to 83.3% (corona-enclosed oocytes; CO) and 54.3% to 88.9% (denuded oocytes; DO), respectively. When DO were cultured for more than 24 h before insemination, fertilization rates were significantly lower compared with CIO and CO. The maximum rates of development to blastocysts were observed when the oocytes were cultured for 24 h in the CIO group (22.1%), 20 h in the CO group (19.7%) and 18 h in the DO group (9.2%), respectively. These results suggest that (i) the presence of cumulus cells or corona cells during maturation is not necessary for nuclear maturation of oocytes; (ii) the attachment of corona cells to the oocytes during maturation is important for the further development to the blastocyst stage, and (iii) the presence of attached cumulus and/or corona cells during maturation in vitro extends the maturation period required for further development to the blastocyst stage.     

Are there indications for intra-cytoplasmic sperm injection in female infertility?

ICSI for oocyte pathology may represent the solution in case of zona pellucida anomalies, deficiency of the oolemma fusion ability or absence of cortical reaction. These indications remain scarce and difficult to be displayed. A link could be shown by some authors between some oocyte morphologic anomalies and IVF fertilization failures which are overcome by ICSI. Other dysmorphic oocytes are associated with an impairment in cytoplasm function which results in a poor embryonic viability on which ICSI has no effect. When few oocytes are recovered (< or = 5) and the semen characteristics are not optimal, ICSI may rectified the decrease in fertilization rates obtained after classical insemination. Lastly, ICSI will become increasingly used in case of oocyte in-vitro maturation or freezing instead of classical IVF which is less efficient.     

Calcium dynamics during starfish oocyte maturation and fertilization

Intracellular free calcium levels in starfish oocytes have been monitored during meiotic maturation and fertilization using calcium-sensitive fluorescent dyes combined with confocal laser scanning microscopy or fura ratioing techniques. In time-lapse analyses of prophase-arrested and maturing oocytes, calcium transients were elicited by inositol 1,4,5-trisphosphate (IP3), ryanodine, or caffeine, indicating that both the IP3-sensitive and IP3-insensitive receptors of the oocyte's calcium release channels could be stimulated to mobilize calcium ions. Fertilization also triggered a global calcium wave that appeared to travel faster around the cortex than through the center of the oocyte, and maturing oocytes developed normally after their fertilization-induced calcium waves had been imaged. Prophase-arrested specimens, on the other hand, did not undergo germinal vesicle breakdown or cleavage after displaying a fertilization-induced calcium transient throughout their cytoplasm and nucleus, confirming previous observations that calcium spikes are not sufficient to induce development in immature oocytes. In addition, although the calcium spikes triggered by sperm or caffeine reached similar normalized peak heights, fertilization-induced calcium waves in maturing oocytes tended to be more prolonged than the fertilization waves observed in prophase-arrested oocytes or the caffeine-triggered spikes elicited at any stage of maturation. Collectively, such findings suggest that the total amount of releasable calcium does not vary appreciably during maturation, but the patterns of the calcium transients can differ depending on the stage of maturation and/or the type of calcium-releasing agent. Possible artifacts affecting these findings are assessed, and the results are discussed relative to the functioning of calcium release pathways during starfish oocyte maturation and fertilization.     

Embryo cloning in cattle: the use of in vitro matured oocytes

In vitro matured (IVM) bovine oocytes were examined to determine their potential viability in embryo cloning. Activation competence, as monitored by pronuclear formation, increased with oocyte age. Oocytes readily formed a pronucleus when challenged with an electrical pulse 30 h after the onset of maturation. Developmental competence of IVM oocytes tended to increase with oocyte age (P = 0.079). Selection of IVM oocytes on the basis of the presence of a polar body 24 h after the onset of maturation and the size of the follicle from which the oocyte was derived improved development of nuclear transfer embryos (polar body positive 25% versus polar body negative 10%, P < 0.05; large follicle oocytes 31% versus small follicle oocytes 14%, P < 0.05). When selected, IVM oocytes were compared with in vivo matured oocytes recovered from superovulated cows and heifers; no difference was detected for the frequency of embryos produced, pregnancies confirmed between days 50 and 60 of gestation, or the number of calves born. We conclude that selected IVM oocytes are equivalent to in vivo matured oocytes when used for bovine embryo cloning.     

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.     

Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C

Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 70-80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3 (final concentration 1 microM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.     

p53 tumor-suppressor gene: clues to molecular carcinogenesis

Pig oocytes were examined to test their ability to undergo cortical granule exocytosis upon penetration by spermatozoa during meiotic maturation. Immature or maturing oocytes (cultured in vitro for 0 h, 26 h and 46 h) were inseminated with ejaculated boar spermatozoa in vitro. Before and after insemination, oocytes were stained with peanut agglutinin labelled with fluorescein isothiocyanate and the cortical granule distributions were examined under the fluorescent microscope and the laser confocal microscope. Before insemination, all the oocytes at the germinal vesicle stage showed a uniform distribution of cortical granules throughout the cortical cytoplasm. The granules migrated centrifugally during maturation and were distributed just beneath the oolemma in the oocytes after germinal vesicle breakdown, forming a monolayer in metaphase I or metaphase II. Cortical granules were still present in all penetrated oocytes at the germinal vesicle stage 18 h after insemination; in contrast, 26% and 84% of the oocytes inseminated at the stages of germinal vesicle breakdown or at metaphase I and II, respectively, completely released their cortical granules. Nuclear activation rates of penetrated oocytes were 0%, 38% and 96% in oocytes cultured for 0 h, 26 h and 46 h, respectively. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 88% (oocytes cultured for 46 h) released cortical granules completely. Complete cortical granule exocytosis was not observed in nuclear-inactivated oocytes. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 80% (oocytes cultured for 46 h) of monospermic oocytes and 67% (oocytes cultured for 26 h) and 91% (oocytes cultured for 46 h) of polyspermic oocytes released cortical granules, and no statistical difference was observed between oocytes cultured for 26 h or 46 h, or between monospermic and polyspermic oocytes. The proportion of oocytes with cortical granule exocytosis increased as insemination time increased and was greatest 18 h after insemination in oocytes cultured for 26 h and 46 h; no obvious changes were observed when the insemination time was prolonged to 24 h. These results indicate that pig oocytes develop the ability to release cortical granules after penetration by spermatozoa following germinal vesicle breakdown, and that this ability is not fully developed until metaphase II. Cortical granule exocytosis is accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic maturation are responsible for the cortical reaction. Polyspermy may be a result of a complete failure of cortical granule exocytosis in immature oocytes and delayed CG exocytosis in matured oocytes.     

Cytokine induction of heat shock protein expression in human oligodendrocytes: an interleukin-1-mediated mechanism

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed.(ABSTRACT TRUNCATED AT 250 WORDS)