Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Protein kinase C and meiotic maturation of surf clam oocytes

We report here that phorbol ester, a potent activator of protein kinase C, induces germinal vesicle breakdown in surf clam oocytes. However, phorbol ester-induced activation is slow and is not accompanied by an increased Ca2+ influx. Simultaneous additions of phorbol ester and various amounts of K+ ions, which induce Ca2+ influx of different amplitudes, result in successful activation within the normal time schedule at K+ concentrations inefficient alone in activating the oocytes. In vivo, increased protein phosphorylation triggered by phorbol ester amounts to about one third that seen after fertilization. These results suggest that increased Ca2+ influx and protein kinase C activation act in synergy to cause resumption of meiotic maturation in these oocytes.     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

Activators of protein kinase C stimulate meiotic maturation of rat oocytes

Agonistic analogs of gonadotropin releasing hormone can induce oocyte maturation in rat follicle-enclosed oocytes (1-5). Cyclic AMP does not rise following exposure of the ovarian follicle to GnRH (3) suggesting that cAMP-dependent protein kinase is not involved in the mechanism of GnRH action in this system. Protein kinase C, which is independent of cAMP, has recently been reported to mediate GnRH action in the pituitary (6-8). The possible involvement of this enzyme in the regulation of oocyte maturation has been tested in the present study. We report here that phospholipase C and direct activators of protein kinase C can mimic the response of rat oocytes to GnRH. These results suggest that GnRH-induced meiotic maturation of rat oocytes is mediated by the phospholipid-dependent protein kinase, protein kinase C.     

Protein kinase C and meiotic regulation in isolated mouse oocytes

In this study, the possible role of protein kinase C (PKC) in mediating both positive and negative actions on meiotic maturation in isolated mouse oocytes has been examined. When cumulus cell-enclosed oocytes (CEO) were cultured for 17-18 hr in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, each of the five different activators and five different antagonists of PKC stimulated germinal vesicle breakdown (GVB) in a dose-dependent fashion. One of the activators, phorbol-12-myristate 13-acetate (PMA), also triggered GVB in CEO arrested with isobutylmethylxanthine or guanosine, but not in those arrested with dibutyryl cyclic AMP. When denuded oocytes (DO) were cultured for 3hr in inhibitor-free medium, all PKC activators suppressed maturation (<10% GVB compared to 94% in controls), while the effect of PKC antagonists was negligible. Four of the five antagonists reversed the meiosis-arresting action of HX in DO. PMA transiently arrested the spontaneous maturation of both CEO and DO, with greater potency in DO. The stimulatory action of PMA in HX-arrested oocytes was dependent on cumulus cells, because meiotic induction occurred in CEO but not DO. PKC activators also preferentially stimulated cumulus expansion when compared to antagonists. A cell-cell coupling assay determined that the action of PMA on oocyte maturation was not due to a loss of metabolic coupling between the oocyte and cumulus oophorus. Finally, Western analysis demonstrated the presence of PKCs alpha, beta1, delta, and eta in both cumulus cells and oocytes, but only PKC epsilon was detected in the cumulus cells. It is concluded that direct activation of PKC in the oocyte suppresses maturation, while stimulation within cumulus cells generates a positive trigger that leads to meiotic resumption.     

Protein kinase C and progesterone-induced maturation in Xenopus oocytes

Though progesterone-induced maturation has been studied extensively in Xenopus oocytes, the mechanism whereby the prophase block arrest is released is not well understood. The current hypothesis suggests that a reduction in cAMP and subsequent inactivation of cAMP-dependent protein kinase is responsible for reentry into the cell cycle. However, several lines of evidence indicate that maturation can be induced without a concomitant reduction in cAMP. We show that the mass of diacylglycerol in whole oocytes and plasma membranes decreases 29% and 10% respectively, within the first 15 sec after the addition of progesterone. Diacylglycerol in plasma membranes further decreased 59% by 5 min. We also show that the protein kinase C inhibitors sphingosine and staurosporine can induce oocyte maturation. In addition, the synthetic diglyceride, DiC8, and microinjected PKC can inhibit or delay progesterone-induced maturation. These results together suggest that a transient decrease in protein kinase C activity may regulate entry into the cell cycle. The mechanism whereby DAG is decreased in response to progesterone is unclear. Initial studies show that progesterone leads to a decrease in IP3 suggesting that progesterone may act by reducing the hydrolysis of PIP2. On the other hand, progesterone caused a decrease in the amount of [3H]arachidonate labelling in DAG during the same time suggesting that progesterone may stimulate lipase activity. The relationship between postulated changes in the PKC pathway and those hypothesized for the PKA pathway are discussed.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

Protein kinase C initially inhibits the induction of meiotic cell division in Xenopus oocytes.

We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and in vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.     

Nuclear maturation of follicle-enclosed rat oocytes during inhibition of steriodogenesis

This study examined the possible role of steriods in meiotic maturation of preovulatory oocytes. Follicles were isolated from PMSG-treated immature rats and incubated with or withour LH in the presence of one of four inhibitors of steroidogenesis. The inhibitors employed had different sites of attack in the steriodogenic pathway and were aminoglutethimide, cyanoketone, SU 10603 (17β-hydroxylase inhibitor), and 4-OH-androstenedione (aromatase inhibitor). As predicted, the inhibitors drastically altered the pattern of steroid production. None of the inhibitors, however, changed the proportion of oocytes resuming or completing meiosis in response to LH, and there was also no effect of the inhibitors on the oocytes in the absence of LH. It was concluded that steriods are not required for preovulatory nuclear maturation of oocytes in the rat.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Involvement of calcium/calmodulin-dependent protein kinase kinase in meiotic maturation of pig oocytes

Calcium (Ca(2+))/calmodulin-dependent protein kinase kinase (CaMKK) is a novel member of Ca(2+)/calmodulin-dependent protein kinase (CaMK) family, whose physiological roles in regulating meiotic cell cycle needs to be determined. We showed by Western blot that CaMKK was expressed in pig oocytes at various maturation stages. Confocal microscopy was employed to observe CaMKK distribution. In oocytes at the germinal vesicle (GV) or prometaphase I (pro-MI) stage, CaMKK was distributed in the nucleus, around the condensed chromatin and the cortex of the cell. At metaphase I (MI) stage, CaMKK was concentrated in the cortex of the cell. After transition to anaphase I or telophase I stage, CaMKK was detected around the separating chromosomes and in the cortex of the cell. At metaphase II (MII) stage, CaMKK was localized to the cortex of the cell, with a thicker area near the first polar body (PB1). Treatment of pig cumulus-enclosed oocytes with STO-609, a membrane-permeable CaMKK inhibitor, resulted in the delay/inhibition of the meiotic resumption and the inhibition of first polar body emission. The correlation between CaMKK and microfilaments during meiotic maturation of pig oocytes was then studied. CaMKK and microfilaments were colocalized from MI to MII during porcine oocyte maturation. After oocytes were treated with STO-609, microfilaments were depolymerized, while in oocytes exposed to cytochalasin B (CB), a microfilament polymerization inhibitor, CaMKK became diffused evenly throughout the cell. These data suggest that CaMKK is involved in regulating the meiotic cell cycle probably by interacting with microfilaments in pig oocytes.     

Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes.

Porcine oocytes cultured in follicular fluid for various periods of up to 48 h were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase activity at metaphase I was approximately 10 times that at the germinal vesicle stage. An abrupt reduction in activity was observed in oocytes emitting the first polar body; then the activity increased again to the same level as at metaphase I. This pattern is similar to those reported in non-mammalian species and supports the concepts that histone H1 kinase is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

Protein kinase activities during maturation in xenopus laevis oocytes

Protein kinase activities have been compared in ovarian oocytes and in ovulated eggs of $\text{Xenopus laeyis}$.In ovaries and ovarian oocytes, we have detected, in addition to an already known (1) cyclic AMP stimulated phosphoprotein kinase, a second very active phosphoprotein kinase which is cAMP-independent.Besides these two activities, a third protein kinase activity becomes detectable after maturation and ovulation: it is a cAMP and cGMP-dependent histone kinase.     

Protein synthesis during hormonally induced meiotic maturation and fertilization in starfish oocytes

Uptake of radioactive amino acids and their incorporation into protein were examined during 1-methyladenine-induced maturation and subsequent fertilization of oocytes of the starfish Patiria miniata. The initial response to the hormone was a nearly immediate decrease in permeability to amino acids, indicating that the site of action of the hormone is on the cell surface. Protein synthesis began to increase starting about 12 min after 1-methyladenine stimulation and prior to germinal vesicle breakdown. It continued to rise throughout the first meiotic division. This protein synthesis was not required for assembly or initial functioning of the meiotic apparatus, although it was necessary for the completion of meiosis. Fertilization had no effect on the rate of protein synthesis.Oocytes of P. miniata provide an example of hormonal stimulation of protein synthesis in an invertebrate system.     

Thapsigargin induces meiotic maturation in surf clam oocytes.

I report here that thapsigargin, an inhibitor of Ca(2+)-ATPase activities in internal Ca2+ stores, induces meiotic maturation in prophase I-arrested surf clam (Spisula solidissima) oocytes. The half-maximal dose for triggering germinal vesicle breakdown (GVBD) is 120 nM. Thapsigargin-induced GVBD is followed by all normal subsequent steps of meiotic maturation including extrusions of first and second polar bodies, with almost normal timing as compared with K(+)-induced activation. Thapsigargin-induced GVBD requires the presence of external Ca2+ at a half-maximal concentration of 0.6 mM. In normal sea water, thapsigargin-induced activation is accompanied by a slightly increased 45Ca2+ uptake by the oocytes and by an intracellular pH rise of 0.3 U. These results show that thapsigargin-sensitive Ca2+ pools regulating Ca2+ fluxes exist in surf clam oocytes, and they also further establish that Ca2+ ions are the major initial trigger for meiosis resumption in this species.     

Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.     

Protein kinase C mediates human neutrophil cytotoxicity

Human neutrophils stimulated with phorbol myristate acetate were able to damage human erythroleukemic K562 cells, in the absence of specific antibody, as assessed by a two hour 51Cr release assay. Neutrophils treated with formyl-peptide fMet-Leu-Phe did not display tumoricidal response, but the addition of diacylglycerol kinase inhibitor R59022 together with formyl-peptide induced the cytotoxic capacity against tumor target cells. Phorbol ester is a potent activator of certain functions of neutrophils because of its ability to directly and irreversibly stimulate protein kinase C; formyl-peptide, on the contrary, activates protein kinase C by inducing a rapid and transient production of diacylglycerol, that is quickly metabolized. The addition of an inhibitor of diacylglycerol kinase, R59022, however potentiated the action of formyl-peptide. These results indicate that protein kinase C is involved in the tumoricidal activity of neutrophils against K562 cells, and that maximal activation of the enzyme is required to achieve the cytotoxic response.     

Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes

The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase- defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD- RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.