Structural changes in bovine oocytes during final maturation in vivo

On the basis of structural observations bovine oocytes were grouped into four successive classed: 0, those before the luteinizing hormone (LH) surge; 1, those up to 8 h following the LH peak level; 2, those between 8 and 19 h after the LH peak level; and 3, those between 19 h after the LH peak level and ovulation. Oocytes in class 0 had mitochondria located in a generally peripheral position. Interior to the mitochondria were elements of rough endoplasmic reticulum (RER) and numerous membrane-bound vesicles which bore ribosome-like particles on their outer surface. The first visible changesater the LH peak level as seen in class 1 were the formation of the periviteline space with loss of contact between the cumulus cells and the oocyte, and ruffing of the nuclear envelope. These changes were followed b the resumption of meiosis as defined by germinal-vesicle breakdown (GVBD), the disappearance of RER, and the formation fo clusters of mitochondria in association with lipid droplets and elementrs of smooth endolasmic reticulum (SER). The period between 8 and 19 h following LH peak level (class 2) was characterized by intensive clustering of mitochoncria in association with lipid droplets and elements of SER, conversion of lipid, fusion of vesicles, and the appearance of ribosomes in the cytoplasm. During the final stage (class 3), the polar body was extruded, the mitochondria dispersed, and the majority of the organelles became located toward the center of the cell. The relatively organelle-free cortical region contained cortical granules immediately adjacent to the plasma membrane together with aggregates of tubular SER. The structural changes are discussed in the context of follicular steroidogenesis and oocyte developmental competence.     

Nuclear maturation of canine oocytes cultured in protein-free media

The objective of this study was to determine the ability of canine oocytes to complete nuclear maturation in a protein-free medium. Oocytes obtained from ovaries of bitches aged 6 months to 2 years were cultured either in TCM199 or CMRL1066 medium without protein supplementation in 5% or 20% O(2). Sixteen of 121 (13%) oocytes cultured in TCM199 reached metaphase II, but only 1 of 135 oocytes cultured in CMRL1066 did so (P < 0.05). Oxygen concentration did not affect nuclear maturation. An additional 103 oocytes were cultured in TCM199 for 48 hr, inseminated with chilled ejaculated spermatozoa, fixed in 1:3 acetic acid-ethanol and then stained with aceto-orcein; 34% of these oocytes were penetrated by spermatozoa. To determine developmental competence of oocytes cultured in a protein-free medium, 85 oocytes were cultured in TCM 199 for 48 hr, inseminated and then cultured; 7 early stage embryos were produced. The effects of growth hormone, beta-mercaptoethanol (betaME), luteinizing hormone (LH) and energy substrates, alone or in combination, on nuclear maturation of oocytes cultured in a protein-free medium were also determined. Growth hormone enhanced cumulus expansion, but did not improve nuclear maturation. beta-mercaptoethanol had no effect on nuclear maturation. However, percentages of MII oocytes significantly decreased when the oocytes were cultured for 48 hr in the medium containing LH or a high concentration of glucose (P < 0.05). In conclusion, canine oocytes are able to complete nuclear maturation in a protein-free medium. The specific type of medium and other supplements significantly influence the meiotic maturation of canine oocytes.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.     

Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes

Based on the morphology and expansion of the cumulus cells, several different classes of porcine cumulus-oocyte complexes (COCs) can be distinguished, during their maturation in vitro. The goal of the present study was to find out the rate of each morphologic category in case of COCs and granulosa-cumulus-oocyte complexes (GCOCs), the characteristics of their nuclear progression, cytoplasmic maturation, and the frequency of monospermy after IVF. It was found that the frequency of cumulus expansion is higher in case of GCOCs than that of COCs. Nuclear progression of COCs was more accelerated than that of GCOCs. Oocytes attached to the bottom of culture dish with dark, compact cumulus underwent nuclear and acquired their ability to be activated earlier than that of oocytes showing normal cumulus expansion. The rate of monospermic fertilization after IVF of normal COCs showing normal cumulus expansion was higher than that of COCs attached to the dish. These results suggest that diverse behavior of cumulus cells during in vitro culture affects nuclear and cytoplasmic maturation of porcine oocytes, which also affects IVF results. It can be concluded that granulosa cells promote normal cumulus expansion thus decrease heterogeneity in nuclear and cytoplasmic maturation amongst oocytes.     

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

Involvement of mitogen-activated protein kinase (MAPK) pathway in LH- and meiosis-activating sterol (MAS)-induced maturation in rat and mouse oocytes

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

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.     

Ultrastructural observations on cortical granules in human follicular oocytes cultured in vitro

The fine structure, distribution, and fate of cortical granules in human oocytes cultured in vitro are reported. Follicular maturation in women with blocked Fallopian tubes was induced by clomiphene citrate and human chorionic gonadotropin, and preovulatory eggs were obtained by improved methods of laproscopy and oocyte recovery. These oocytes were then inseminated and cultured in a modified Ham's F10 medium for 3 to 72 hr to assess their fertilizability. Cortical granules were observed in all 17 unfertilized oocytes investigated, which had completed various stages of meiotic maturation. A marked increase in their numbers was observed in oocytes cultured for 3 to 6 hr. There was no evidence of spontaneous cortical granule release in any of the oocytes studied. It is concluded that cortical maturation expressed by proliferation of cortical granules is as significant a criterion as nuclear maturation in assessing maturity and fertilizability of oocytes cultured in vitro. A short sojourn in culture before insemination could improve chances of normal fertilization and embryo development, which has been recently achieved in our laboratory.     

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig

In mammals, the nucleolus of full‐grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full‐grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non‐treated or actinomycin D‐treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re‐injection of nucleoli from growing oocytes (23%), but not when nucleoli from full‐grown oocytes were re‐injected into enucleolated, growing oocytes (49%). When enucleolated, full‐grown oocytes were injected with nucleoli from growing or full‐grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full‐grown oocytes injected with nucleoli from full‐grown oocytes matured to metaphase II (56%), whereas injection with growing‐oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing‐oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full‐grown oocyte nucleolus has lost the ability. Mol. Reprod. Dev. 78:426–435, 2011. © 2011 Wiley‐Liss, Inc.     

Nuclear ultrastructure in bovine oocytes after inhibition of meiosis by chemical and biological inhibitors

Various components of the ovarian follicle as well as different chemicals can suppress the resumption of meiosis in cumulus-oocyte complexes (COCs). In this study the nuclear ultrastructure of bovine COCs was assessed after 8 h of meiotic inhibition with 50 microM roscovitine (ROSC), 50 microM butyrolactone (BL-I), 2 mM 6-DMAP, 2 microM cycloheximide (CX), or a theca cell monolayer (TC). COCs were recovered according to standard in vitro methods, cultured in a simple and defined medium, and processed for transmission electron microscopy. Control COCs were processed before onset of culture and multiple oocytes were evaluated for each treatment. In all groups, the oocyte nucleus presented a dense fibrillar nucleolus consisting of a fibrillar sphere with a fibrillar center. In TC and 6-DMAP inhibited COCs condensed chromatin adhered to the nucleolus while in all other groups the perinuclear chromatin was separated from the nucleolus. In ROSC inhibited COCs, the nuclear envelope presented only slight small amplitude undulation. The BL-I-inhibited COCs presented an intermediate level of low amplitude undulation of the NE. In CX, 6-DMAP, and TC inhibited COCs the nuclear envelope presented extensively low amplitude undulations. In ROSC inhibited COCs, electron-dense granules formed ring-shaped structures. In some of the BL-I inhibited COCs multiple stellate crystal-like structures were found, and in these COCs the nuclear envelope and the perinuclear cisternae appeared less distinct than in the other BL-I inhibited COCs. In 6-DMAP inhibited COCs interchromatin-like granule clusters were present. In conclusion, the oocyte nuclei in all COCs presented a dense fibrillar nucleolus resembling that in control COCs. However, variations were observed in 1) the nuclear envelope morphology; 2) the chromatin location in relation to the nucleolus; and 3) the presence of different populations of intranuclear granules. Although all treatments inhibited oocyte nucleus breakdown, the mechanisms underlying these effects are different and require further characterization. Mol. Reprod. Dev. 59: 459-467, 2001.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.     

Fate of the first polar bodies in mouse oocytes

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

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Effects of NO-synthase inhibitors on maturation mouse oocytes in cumulus-oocyte complexes

We studied the effects of various NO-synthase inhibitors on meiotic maturation of mouse oocytes in vitro in cumulus-oocyte complexes isolated from follicles of varying sizes. Selective and nonselective inhibitors of NO-synthase isoforms suppressed meiotic maturation of oocytes to varying degrees, which was expressed in a decreased number of oocytes at metaphase II. The results obtained suggest that the role of inducible form of NO-synthase (iNOS) increases with the development of follicles and oocytes.