Effect of defolliculation and 17α-hydroxy, 20β-dihydroprogesterone on cyclic AMP level in full-grown oocytes of the rainbow trout,Salmo gairdneri

The changes in cAMP were followed in trout oocytes incubated in vitro after defolliculation performed by either enzymatic or manual dissection. Both defolliculation methods induced a highly significant rise in oocyte cAMP level (4.5 times the basal level of control [follicle-enclosed oocytes], after 6 h). Treatment of defolliculated oocytes with 17α-hydroxy,20β-dihydroprogesterone (17α,20β-OH-P) (10^?6 M), which induced oocyte maturation (germinal vesicle breakdown [GVBD]) was able, first, to interrupt the increase of oocyte cAMP level promoted by defolliculation and then to lower this level significantly down to values that still remained higher than folliculated controls. Very low concentrations of 17α,20β-OH-P (1.38–55.6 10^?9 M), or physiological doses of testosterone (0.35 10^?6 M, in the range found in vivo before ovulation) were able to induce a similar decrease of oocyte cAMP level without inducing GVBD. Under the same experimental conditions estradiol (0.35 10^?6 M) exhibited no action. These results suggest that some factor(s) originating in the follicle (FIF), inhibit the oocytes' tendency to accumulate cAMP before the final surge of 17α,20β-OH-P. This factor might be a follicular steroid such as testosterone or nonmaturing concentrations of 17α,20β-OH-P. Moreover our data favour the hypothesis that the final surge of 17α,20β-OH-P could induce distinct intraoocyte mechanisms: the first induces an irreversible blockage of cAMP level before the inhibitory action of the FIF is suppressed by ovulation, and the second mechanism leads to GVBD.     

Intracellular pH Increase Driven by an Na+/H+ Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pH_i) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pH_iof 0.3 pH unit after activation by excess K⁺. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pH_iwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K⁺induced GVBD but the pH_iof K⁺-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pH_iincreases in K⁺-activated oocytes were otherwise proportional to the external Na⁺concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K⁺-induced increase of pH_ibut did not block GVBD. These two derivatives were able, however, to retard K⁺-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K⁺-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pH_iincrease, driven by a typical Na⁺/H⁺exchanger, follows activation of surf clam oocytes but that this pH_iincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.     

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.     

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K⁺-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with ³⁶S-methionine or ³²P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K⁺-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K⁺ or serotonin (12–15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes. © 1996 Wiley-Liss, Inc.     

Intracellular pH change does not accompany egg activation in the mouse

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pH_i). We measured pH_i, in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH_i was 6.96 ± 0.004 (± SEM) in GV-intact oocytes, 7.00 ± 0.01 in ovulated, unfertilized eggs, and 7.02 ± 0.01 in fertilized zygotes, indicating no sustained changes in pH_i after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pH_i occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pH_i followed ethanol activation. Since increased Na⁺/H⁺ antiporter activity is responsible for pH_i increase in the sea urchin, pH_i was measured in the absence of added bicarbonate or CO₂ la condition under which the antiporter would be the only major pH_i regulatory mechanism able to operate, since the others were bicarbonate- dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na⁺/H⁺ antiporter was activated. There was no physiologically significant difference in pH_i after GVBD or fertilization, when pH_i was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pH_i is not a feature of egg activation in the mouse. © 1996 Wiley-Liss, Inc.     

Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes

Full-grown Xenopus oocytes undergo meiotic maturation in response to progesterone stimulation. Using [¹⁴C]dimethyloxazolidine dione (DMO), we have measured a cytoplasmic alkalization in these oocytes starting at pH 7.14 ± 0.17 during the germinal vesicle (GV) stage, and increasing to 7.56 ± 0.14 at the time of germinal vesicle breakdown (GVBD). During this period, the rate of protein synthesis increases 2-fold from 18.9 ± 3.1 to 37.7 ± 8.8 ng/hr/oocyte. Artificial alkalization of GV stage oocytes to pH_i 7.68 ± 0.16, by exposure to the weak bases trimethylamine, methylamine, procaine, or imidazole, led to a 1.8-fold increase in the synthetic rate. Intracellular acidification from 7.5 back to 7.0 had no apparent effect on the elevated rate of protein synthesis following GVBD. Therefore, a cytoplasmic alkalization in the range of 7.5 to 7.6 seems to be one of the events that is necessary for initiating the increase in protein synthesis in maturing Xenopus oocytes; however, it does not appear that an elevated pH_i is necessary to maintain the increased synthetic rate following GVBD.     

Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.     

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.     

Signaling pathways in ascidian oocyte maturation: the role of cyclic AMP and follicle cells in germinal vesicle breakdown

Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 n m in a reversible manner.     

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP3) releases stored intracellular Ca2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4-7.0 x 10(-21) moles of InsP3 (equivalent to 5-80 pM if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP3 can mimic part of the activation process in this protostome. Synthesis of InsP3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP2 is most likely due to its hydrolysis, yielding InsP3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.     

Towards a new understanding on the regulation of mammalian oocyte meiosis resumption

Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in granulosa cells in oocyte meiosis resumption. Unresolved questions and future directions on research regarding signaling changes in follicle cells and oocytes as well their communication in response to the gonadotropin surge are addressed in this review.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l-methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy. Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage-dependent sodium channels, without altering the other main conductances, Cl^?, K⁺ or Ca²⁺. After such a treatment, the relative permeability of the membrane to Na⁺ as compared to K⁺ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced. These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm-gated channels which are supposed to trigger the fertilization potential.     

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.     

Fast polyspermy block and activation potential : Electrophysiological bases for their changes during oocyte maturation of a starfish

In the starfish oocyte, the activation potential (AVP) upon fertilization establishes a fast polyspermy block. In the present paper, factors affecting the peak level of the AVP were analyzed by comparing current-voltage relations of the oocyte membrane just before and after insemination at various maturation stages. These factors were: (1) conductance of the oocyte membrane before insemination; (2) magnitude of the conductance increase induced by sperm; and (3) equilibrium potential of the AVP. Mature oocytes showed extremely low membrane conductance before insemination, and this provided a more positive-going AVP establishing monospermy. Overmature oocytes before fertilization showed higher conductance than mature oocytes and usually became polyspermic upon insemination. Application of Ba²⁺ ions reduced the conductance of the unfertilized, overmature oocyte to a state similar to that of the mature oocyte. Correspondingly, Ba²⁺ reduced the probability of polyspermy in overmature oocytes. Old oocytes showed even higher conductance before insemination than overmature oocytes and, in addition, apparently showed a large negative value for the equilibrium potential of the AVP. In old oocytes, these two factors account for the small amplitude of the AVP, which allows multiple sperm entries. Furthermore, the magnitude of the conductance increase induced by sperm seemed to change during the maturation process.     

The effects of quercetin on meiosis initiation in clam and starfish oocytes

We have treated Spisula and Asterias oocytes with quercetin to determine the effects of this drug on germinal vesicle breakdown (GVBD). Quercetin (100-200 microM) reversibly inhibited GVBD when induced by excess KCl or ionophore A23187. Kinetic studies showed that quercetin blocked an early event in GVBD. Lower concentrations of quercetin (10-20 microM) blocked fertilization. However, quercetin sensitized the oocytes to initiation of GVBD by excess ions which do not normally trigger GVBD. Quercetin (100-200 microM) also blocked 1-methyladenine-induced GVBD in the starfish. In subthreshold concentrations of the hormone or in its absence, lower concentrations (20-40 microM) stimulated GVBD. The results support the hypothesis that quercetin exerts its effects on meiosis initiation through its effects on calcium sequestration.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Timing of final oocyte maturation in Acropora and merulinid corals

Final oocyte maturation is a gametogenic process that occurs at the end of oogenesis, which includes germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD). This process is essential for oocytes to become competent for fertilization and occurs just before spawning. Timing of GVM and GVBD has been extensively studied to understand reproductive timing in various marine organisms. For corals, however, such information remains scarce. Here, we examined the timing of GVM and GVBD in broadcast-spawning corals: two Acropora and six merulinid species, from around the full moon until spawning day(s), at Lyudao, Taiwan in 2019 and 2020. The proportion of oocytes that had completed GVM around the full moon varied among species (14–64%), while the proportion increased to > 80% by 6–7 d after the full moon when many sampled colonies spawned. By contrast, although data are limited, the timing of GVBD indicated a more uniform pattern among species, and the onset of GVBD in the majority of oocytes occurred within 3–10 h before spawning. As GVM and GVBD are prerequisites for fertilization, and probably the spawning itself, the interspecific variation in the timing of GVM likely reflects an interspecies variation in spawning timing. This hypothesis may partially explain the different spawning timing (days) among coral species observed at the study location. Further research is needed to test such a hypothesis, given the limitation of our study with regard to the number of observations, annual variation, and examined coral taxa.

     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.