In vitro inhibition of oocyte and follicular maturation and spawning in starfish (Asterias forbesi) by 2-4-dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of 3H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

In vitro Inhibition of Oocyte and Follicular Maturation and Spawning in Starfish (Asterias forbesi) by 2-4-Dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of ³H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

Properties of 1-methyladenine receptors in starfish oocyte membranes: involvement of pertussis toxin-sensitive GTP-binding protein in the receptor-mediated signal transduction.

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a guanine nucleotide-binding protein (G protein) serving as the substrate of pertussis toxin is involved in the 1-MA receptor-mediated signal. We thus investigated properties of 1-MA receptors by means of binding of the radiolabeled ligand to the oocyte membranes. There were apparently two forms of 1-MA receptors with high and low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. A 39-kDa protein, which had been identified as the alpha-subunit of the major substrate G protein for pertussis toxin, was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the 39-kDa pertussis toxin-substrate G protein in starfish oocyte membranes.     

Maturation-Inducing Substances in Asteroid and Echinoid Oocytes

In starfish a hormonal peptide, gonad-stimulating substance(GSS), which is released from nervous tissue, acts on the gonadto produce a maturation-inducing substance (MIS), an inducerof oocyte maturation and spawning. MIS is 1-methyladenine (1-MA).This substance acts on the surface of the oocyte. Cytoplasmicmaturation as revealed by fertilizability is also induced by1-MA. The amount of 1-MA can be determined very accurately witha bioassay method using isolated oocytes. In 1-MA formation,GSS seems to enhance the methylation of some compound whichcontains a purine nucleus at its N1 site. The methyl donor isprobably S-adenosylmethionine. 1-Methylated precursor seemsto be transformed to 1-methyl AMP and then hydrolyzed into 1-methyladenosineand phosphate by phosphomonoesterase. 1-Methyladenosine is finallysplit into 1-MA and ribose by 1-methyladenosine ribohydrolase.So-called spontaneous maturation of oocytes isolated in seawater is due to the action of 1-MA produced in follicle cellseven in the absence of GSS. 1-MA is present in echinoid gonadsand seems to act as MIS also in these animals. Disulfidereducingagents such as dithiothreitol and 2,3-dimercapto-1-propanolinduce starfish oocyte maturation. On the other hand, sulfhydrylreagents such as p-chloromercuribenzoate, iodoacetamide, andN-ethylmaleimide suppressed 1-MA-induced oocyte maturation.Since Concanavalin A acts on the follicle cells to produce 1-MA,the action of this substance seems to be quite similar to thatof GSS.     

腺嘌呤对体外小鼠卵母细胞减数分裂的抑制

本文研究了嘌呤类物质对小鼠卵母细胞减数分裂的影响。于卵母细胞的生发泡内显微注射腺嘌呤和腺嘌呤的类似物苄基腺嘌呤可显著抑制卵母细胞的分裂的重新启动。同时发现在腺嘌呤的作用过程中,腺苷酸环化酶的激活剂氟化钠可增强其对卵母细胞的抑制作用,表明cAMP途径在小鼠卵母细胞减数分裂成熟过程中起重要作用。腺嘌呤在不同培养液中的抑制效果不一,次黄嘌呤在DMEM和EMEM中对小鼠的卵丘细胞-卵母细胞复合体(COC)和无卵丘细胞的裸卵(DO)均具有明显的抑制效应。但腺嘌呤在DMEM比在EMEM中对COC的抑制效果更强,而且腺嘌呤在DMEM中与次黄嘌呤具有协同效应,这些差别可能是由于两种培养液中不同成分如谷氨酰胺造成卵母细胞对腺嘌呤吸收差异而引起的。     

SITE OF 1-METHYLADENINE RECEPTORS IN THE MATURATION OF STARFISH OOCYTES

Maturation of vitelline coat-free (VCF) oocytes of the starfish, Asterina pectinifera , was studied. When the oocytes, the vitelline coats of which were elevated by adding the ionophorc A-23187, were forced through two sheets of copper mesh, the vitelline coats were completely removed from the oocytes. Although some of the VCF oocytes underwent germinal vesicle breakdown following this mechanical treatment, most of them retained the normal germinal vesicles. These VCF immature oocytes underwent breakdown of germinal vesicles after addition of 1-methyladenine (1-MA). Dose-response curves of VCF oocytes to 1-MA were similar to those of normal oocytes. These results indicate that 1-MA reacts with the plasma membrane and that the presence of the vitelline coat is not prerequisite for inducing oocyte maturation.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.     

Germinal vesicle contents are required for the cytoplasmic cycle during meiotic division of starfish oocytes

Enucleated oocytes of starfish still show cyclic changes in cortical tension with a temporal pattern similar to that exhibited by intact oocytes during meiotic division, provided that the enucleation is performed a certain time after the breakdown of the germinal vesicle (K. Yamamoto and M. Yoneda, Dev. Biol. 96, 166-172, 1983). If an oocyte is bisected immediately after germinal vesicle breakdown, the resulting nonnucleate fragment shows some change in tension, but the pattern of change is much less regular than that seen in intact oocytes, suggesting that the dispersion of germinal vesicle (GV) contents into cytoplasm is required for the establishment of the cytoplasmic cycle. In order to demonstrate the role of GV contents directly, nonnucleate fragments derived from immature oocytes were injected with GV contents taken from other immature oocytes. On treatment with 1-methyladenine (1-MA) these fragments showed two rounds of increase in tension as is characteristic of intact maturing oocytes. The first rise in tension was always observed 50-70 min after the treatment with 1-MA, similar to the time of first polar body formation in intact oocytes, regardless of the time of injection of GV contents. Even when GV contents were injected into nonnucleate fragments which had been already treated with 1-MA, these fragments showed two rounds of change in tension. The timing of the first rise in tension was found to be 38 +/- 7 min after injection, irrespective of the time of the foregoing treatment with 1-MA. These results prove the indispensability of GV contents for inducing the cytoplasm of the maturing starfish oocyte to initiate its own cyclic activity, and suggest that the normal process of cytoplasmic maturation may consist of two phases, i.e., (1) a GV-independent phase initiated by 1-MA treatment, and (2) a second phase initiated by mixing of GV contents with cytoplasm.     

Two-stage dependence for 1-methyladenine induced reinitiation of meiotic maturation in starfish oocytes

The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to ≥ 0.5 µM 1-MA causes germinal vesicle breakdown (GVBD) in ∼ 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (∼ 10 min) when they are exposed to 1 µM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 µM 1-MA for up to 10 min followed by 1 µM 1-MA is equivalent to continuous exposure to 1 µM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.     

Maturation hormone induced an increase in the translational activity of starfish oocytes coincident with the phosphorylation of the mRNA cap binding protein,eIF-4E,and the activation of several kinases

The stimulation of translation in starfish oocytes by the maturation hormone, 1-methyladenine (1-MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF-4F, and the guanine nucleotide exchange factor for eIF-2, eIF-2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish, Pisaster orchraceus. Increased phosphorylation of eIF-4E, the cap binding subunit of the eIF-4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF-4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen-activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF-4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF-4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12-myristate 13-acetate, an activator of protein kinase C, for 30 min prior to the addition of 1-MA resulted in the inhibition of 1-MA-induced phosphorylation of eIF-4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF-4E, after very early events of maturation. Another possibility is that eIF-4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1-MA. In conclusion, our results suggest a role for the phosphorylation of eIF-4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth in mammalian cells. © 1993 Wiley-Liss, Inc.     

Oocyte maturation is inhibited by adenosine in the presence of follicle-stimulating hormone

Considerable evidence implicates cyclic 3', 5' adenosine monophosphate (AMP) in the maintenance of meiotic arrest of mammalian oocytes. Since this laboratory previously found that adenosine augmented follicle-stimulating hormone (FSH)-stimulated accumulation of cyclic AMP in oocyte-cumulus-complexes (OCC), in the present studies we investigated the possibility that adenosine inhibits maturation of oocytes. In rat OCC cultured in the presence of FSH, adenosine markedly inhibited oocyte maturation in a dose-dependent and biphasic manner. Maximum inhibition of oocyte maturation was seen with 1-30 microM adenosine in the presence of FSH, and half-maximal inhibition occurred with less than 0.3 microM adenosine. High levels of adenosine (100 microM) did not inhibit oocyte maturation in the presence of FSH. In the absence of FSH, adenosine showed little effect on oocyte maturation in the present studies, but increased the maximum inhibition of oocyte maturation produced by FSH approximately twofold. Like adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine 5'-monophosphate (AMP) also inhibited oocyte maturation; whereas adenine, guanosine, inosine, and hypoxanthine were inactive at equivalent levels. The metabolism-resistant adenosine analog (2-chloroadenosine) was as active an inhibitor as adenosine. Inhibition produced by the adenine nucleotides may have been direct or due to conversion to adenosine by extracellular nucleotidases. The concentration dependence and purine specificity for inhibition of oocyte maturation are characteristic of an adenosine receptor-mediated process, but direct evidence for such a mechanism was not shown. The effective concentration of adenosine for inhibition of oocyte maturation is within the range of reported levels of adenosine in biological tissues and fluids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interference with interaction between eukaryotic translation initiation factor 4G and poly(A)-binding protein in Xenopus oocytes leads to inhibition of polyadenylated mRNA translation and oocyte maturation.

The interaction between eukaryotic translation initiation factor 4G (eIF4G) and the poly(A)-binding protein (PABP) facilitates translational initiation of polyadenylated mRNAs. It was shown recently that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces polyadenylated mRNA translation and dramatically inhibits progesterone-induced oocyte maturation. These results strongly suggest that the eIF4G-PABP interaction plays a critical role in the translational control of maternal mRNAs during oocyte maturation. In the present work, we employed another strategy to interfere eIF4G-PABP interaction in Xenopus oocytes. The amino-terminal part of eIF4GI containing the PABP-binding site (4GNt-M1) was expressed in Xenopus oocytes. 4GNt-M1 could bind to PABP in oocytes, which suggests that 4GNt-M1 may evict PABP from the endogenous eIF4G. The expression of 4GNt-M1 resulted in reduction of polyadenylated mRNA translation. Furthermore, 4GNt-M1 inhibited progesterone-induced oocyte maturation. In contrast, 4GNt-M2, in which the PABP-binding sequences were mutated to abolish the PABP-binding activity, could not inhibit polyadenylated mRNA translation or oocyte maturation. These results further support the idea that the eIF4G-PABP interaction is critical for translational regulation of maternal mRNAs in oocytes.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.     

Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.     

Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5 degrees C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5 degrees C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41 degrees C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.     

A receptor linked to a Gi-family G-protein functions in initiating oocyte maturation in starfish but not frogs

The stimulation of oocyte maturation by 1-methyladenine in starfish, and by a steroid in frogs, has been proposed to involve G-protein-coupled receptors. To examine whether activation of receptors linked to G(i) or G(z) was sufficient to cause oocyte maturation, we expressed mammalian G(i)- and G(z)-linked receptors in starfish and frog oocytes. Application of the corresponding agonists caused meiosis to resume in the starfish but not the frog oocytes. We confirmed that the receptors were effectively expressed in the frog oocytes by using a chimeric G-protein, G(qi), that converts input from G(i)- and G(z)-linked receptors to a G(q) output and results in a contraction of the oocyte's pigment. These results argue against G(i) or G(z) functioning to cause maturation in frog oocytes. Consistently, maturation-inducing steroids did not cause pigment contraction in frog oocytes expressing G(qi), and G(z) protein was not detectable in frog oocytes. For starfish oocytes, however, our results support the conclusion that G(i) functions in 1-methyladenine signaling and suggest the possibility of using frog oocyte pigment contraction as an assay to identify the 1-methyladenine receptor. To test this concept, we coexpressed G(qi) and a starfish adenosine receptor in frog oocytes and showed that applying adenosine caused pigment contraction.