Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.     

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Stimulators of AMP‐activated kinase (AMPK) inhibit seawater‐ but not cAMP‐induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling

Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (=“germinal vesicle breakdown”, GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP‐activated kinase (AMPK), a well‐documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up‐ or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW‐ or cAMP‐induced GVBD, given that the catalytic α‐subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK‐mediated phosphorylation of acetyl‐CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice‐cold calcium‐free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW‐ or SW‐solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD‐regulating mechanism involving AMPK deactivation by cAMP‐mediated S485/491 phosphorylation. Mol. Reprod. Dev. 77: 497–510, 2010. © 2010 Wiley‐Liss, Inc.     

Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms II. The roles of tyrosine kinases and phosphatases

Instead of blocking oocyte maturation as it does in most animals, cAMP causes oocytes of marine nemertean worms to initiate maturation (=germinal vesicle breakdown, "GVBD"). To characterize cAMP-induced GVBD in nemerteans, inhibitors of tyrosine kinase signaling were tested on Cerebratulus sp. oocytes that had been incubated in cAMP-elevating drugs versus seawater (SW) alone. Such tests yielded similar results for Src-like tyrosine kinase blockers, as the inhibitors prevented mitogen-activated protein kinase (MAPK) activation without stopping either GVBD or maturation-promoting factor (MPF) activation in both SW and cAMP-elevating treatments. Alternatively, genistein, a general tyrosine kinase antagonist, and piceatannol, an inhibitor of the tyrosine kinase Syk, reduced GVBD and MAPK/MPF activities in SW-, but not cAMP-induced maturation. Similarly, inhibitors of the human epidermal growth factor receptor-2 (HER-2) tyrosine kinase prevented GVBD and MAPK/MPF activations in oocytes treated with SW, but not with cAMP-elevating drugs. Antagonists of either protein tyrosine phosphatases (PTPs) or the dual-specificity phosphatase Cdc25 also reduced GVBD and MAPK/MPF activities in SW-treated oocytes without generally affecting cAMP-induced maturation. Collectively, these data suggest cAMP triggers GVBD via pathways that do not require MAPK activation or several components of tyrosine kinase signaling. In addition, such differences in tyrosine kinase cascades, coupled with the dissimilar patterns of Ser/Thr kinase signaling described in the accompanying study, indicate that nemertean oocytes are capable of utilizing multiple mechanisms to activate MPF during GVBD.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.     

Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms I. The roles of serine/threonine kinases and phosphatases

Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD.     

Interactions between mitogen‐activated protein kinase and protein kinase C signaling during oocyte maturation and fertilization in a marine worm

In the marine nemertean worm Cerebratulus, follicle‐free oocytes re‐initiate meiosis and undergo nuclear disassembly (=germinal vesicle breakdown, GVBD) after being stimulated to mature by seawater (SW) or cAMP‐elevating drugs. Previously, it has been shown that inhibitors of mitogen‐activated protein kinase (MAPK) or protein kinase C (PKC) signaling can reduce SW‐induced GVBD in nemertean oocytes without affecting cAMP‐induced GVBD. Thus, SW and cAMP elevators may trigger alternative pathways that vary in their dependence on MAPK and PKC. To further characterize such signaling cascades, immunoblotting analyses of MAPK and PKC activities were conducted on oocytes treated with U0126, an inhibitor of the MAPK kinase (MAPKK) that is responsible for activating MAPK. Based on these analyses and comparisons with the MAPKK inhibitor CI1040 that inactivates MAPK without preventing GVBD, U0126 seems to block GVBD via a non‐MAPK‐mediated effect that involves PKC. Moreover, evidence is presented for post‐GVBD oocytes establishing positive feedback between MAPK and PKC signaling. Such feedback apparently allows the activities of both kinases to be maintained before insemination and to undergo concomitant downregulation after fertilization. Furthermore, in oocytes treated with MAPKK and PKC inhibitors during fertilization, sperm incorporation and polar body formation still occur, but normal cleavage is prevented. This suggests that although GVBD and aspects of post‐fertilization activation may proceed in the absence of MAPK or PKC, such kinases are apparently required for proper embryogenesis. Collectively, these results are discussed relative to previous analyses of the interactions and functions of MAPK and PKC signaling during oocyte maturation and fertilization. Mol. Reprod. Dev. 76: 708–721, 2009. © 2009 Wiley‐Liss, Inc.     

Roles of protein kinase C isotypes during seawater‐versus cAMP‐induced oocyte maturation in a marine worm

Based on immunoblotting analyses using phospho‐specific antibodies, follicle‐free oocytes of the marine nemertean worm Cerebratulus sp. activate protein kinase C (PKC) when induced to mature by either seawater (SW) or cAMP‐elevating drugs. In SW‐stimulated oocytes, the onset of maturation (=germinal vesicle breakdown, “GVBD”) can be inhibited by broadly acting PKC antagonists such as bisindoylmaleimide (BIM)‐I or BIM‐IX. Conversely, co‐treatment with SW solutions of BIM‐I or BIM‐IX plus a cAMP elevator (forskolin, serotonin, or a phosphodiesterase inhibitor) restores GVBD, indicating that the blockage of SW‐induced GVBD by PKC antagonists is not simply due to oocyte morbidity and that such inhibition is somehow reversible by cAMP signaling. In tests to determine which specific PKC may be involved in regulating GVBD, immunoblots fail to provide strong evidence for the presence of conventional or novel PKCs, which are characteristically activated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Moreover, inhibitors of TPA‐sensitive PKCs do not prevent SW‐induced GVBD, and TPA itself serves to downregulate, rather than stimulate, GVBD. Alternatively, maturing oocytes apparently possess phosphorylated forms of TPA‐insensitive isotypes, including an ～67‐kDa atypical PKC and an ～130‐kDa PKC‐related kinase (PRK). Accordingly, inhibitors of atypical PKC signaling block SW‐but not cAMP‐induced GVBD, collectively suggesting that instead of depending on a conventional or novel isotype, SW‐induced GVBD may require atypical PKC and/or PRK. In addition, such findings provide further support for the view that GVBD in nemertean oocytes can be achieved via multiple mechanisms, with SW triggering different signaling pathways than are stimulated in the presence of cAMP‐elevating drugs. Mol. Reprod. Dev. 76: 693–707, 2009. © 2008 Wiley‐Liss, Inc.     

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10?μM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10?μM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.     

Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed.     

Involvement of cAMP in initiating maturation of the brittle-star Amphipholis kochii oocytes: induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterase and activators of adenylate cyclase

The maturation of brittle-star (Amphipholis kochii) oocytes, i.e., the reinitiation of meiosis accompanied by germinal vesicle breakdown (GVBD) and the acquisition of fertilizability, was induced by acid (pH 3.0) seawater containing 10 mM cAMP. Oocyte maturation was also induced by seawater of normal pH (pH 8.0) that contained either an inhibitor of cyclic nucleotide phosphodiesterase (25 mM theophylline, 25 mM caffeine) or an activator of adenylate cyclase (100 microM forskolin, 0.6 microM cholera toxin). Experiments in which the oocytes were treated with forskolin or theophylline for various periods of time demonstrated that there was a positive correlation between the oocyte cAMP level measured by radioimmunoassay and the extent of GVBD induced in each treatment: both increased as the treatment period became longer and about a threefold increase in cAMP level induced 50% GVBD. These results indicate that an increase in cAMP level initiates maturation of the brittle-star oocytes.     

Pharmacology of the serotonergic inhibition of steroid-induced reinitiation of oocyte meiosis in the teleost Fundulus heteroclitus

Serotonin (5-HT) was found to inhibit steroid (17α,20β-dihydroxy-4-pregnen-3-one; 17,20βP)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > α-methyl-5-HT > 2-methyl-5-HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20βP-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT_1A, 5-HT₂, and 5-HT₃ receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs. Mol. Reprod. Dev. 48:282–291, 1997. © 1997 Wiley-Liss, Inc.     

The role of RhoA in the germinal vesicle breakdown of mouse oocytes

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.     

Comparative biology of oogenesis in nemertean worms

Stricker, S. A., Smythe, T. L., Miller, L. and Norenburg, J. L. 2001. Comparative biology of oogenesis in nemertean worms. — Acta Zoologica (Stockholm) 82 : 213–230
In order to supplement previous analyses of oogenesis in nemertean worms, this study uses light and electron microscopy to compare the ovaries and oocytes in 16 species of nemerteans that represent various taxa within the phylum. Nemertean ovaries comprise serially repeated sacs with an ovarian wall that characteristically includes myofilament-containing cells interspersed among the germinal epithelium. Each oocyte can attach to the germinal epithelium by a vegetally situated stalk and resides in the ovarian lumen without being surrounded by follicle cells. In the ovary, oocytes arrest at prophase I of meiosis and contain a hypertrophied nucleus ('germinal vesicle') that often possesses multiple nucleoli. Intraovarian growth apparently involves an autosynthetic mode of yolk formation in most nemerteans and generates oocytes that measure ~60 µm to 1 mm. When fully developed, oocytes can be discharged through a short gonoduct and are either spawned freely or deposited within egg cases. In most species, oocytes released from the ovary possess extracellular coats and resume maturation by undergoing germinal vesicle breakdown (GVBD). Such post-GVBD specimens also form a punctate endoplasmic reticulum that may facilitate fertilization and development.     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.     

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.     

A reversible, hydrogen ion blockade of spontaneous oocyte maturation in the starfish: locus of action.

Starfish (Asterias forbesi) oocytes encased within their follicle cells mature spontaneously during a portion of the normal reproductive period when released from the ovary into seawater. A previous report has shown that oocytes isolated in acidic seawater do not mature spontaneously but retain the capacity to do so when returned to normal seawater. The object of this study was to determine the mechanism by which acidic pH reversibly blocks spontaneous oocyte maturation in isolated follicles. Incidence of spontaneous oocyte maturation in follicles isolated in acidic seawater decreased as pH decreased from 7 to 4. Oocytes in which spontaneous maturation was inhibited (ASW at pH 4.7 TO 5.4) underwent germinal vesicle breakdown with the addition of 1-methyladenine. Oocytes isolated in acidic seawater (pH 4 or 5) with intact follicle cells matured spontaneously when transferred immediately to normal seawater pH 8); after four hours, 60-65% of the follicles incubated in seawater at pH 5 matured spontaneously when returned to normal seawater as compared to less than 10% of the follicles maintained at pH 4. Inhibition of spontaneous maturation was not reversible in the absence of the follicle cells. Oocytes isolated in acidic seawater with their follicle cells did not spontaneously mature when transferred to calcium-free seawater at pH 8. The results obtained support the hypothesis that acidic seawater reversibly inhibits spontaneous oocyte maturation by interfering with the release of meiosis-inducing substance from the follicle cells.     

Concanavalin A induces germinal vesicle breakdown in Barnea candida (Mollusca,Pelecypoda) oocytes

Treatment of Barnea candida oocytes with 5 μg/ml Con A or above elicits germinal vesicle breakdown (GVBD), the timing for this event being dose dependent. At 100 μg/ml, GVBD occurs within 20 to 30 min, a lag time corresponding to that observed after fertilization. Con A-induced GVBD requires the presence of 2 mM external calcium during all the treatment period while at 10 mM external Ca²⁺, the calcium-dependent period is slightly reduced. It is sensitive to low pH Na-acetate sea water, 50 μM trifluoperazine, 20μM D-600 or 2,4-dinitrophenol, as well as to 10 μg/ml cytochalasin B. A straightforward interpretation of these data would be that Con A-induced maturation is sustained by an energy-requiring effector mechanism involving intracellular contractile proteins.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.