Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus

The effect in vitro of oocyte maturation inhibitors and the ability of preparations of luteinizing hormone to relieve the arrest induced by these substances was studied in preparations of cumulus-free (naked) rat oocytes, and compared to previously obtained results from oocytes enclosed by their cumulus cells. The development of both the cumulus-oocyte complex and naked oocyte is arrested in vitro by cyclic AMP derivatives or cyclic nucleotide phosphodiesterase inhibitors. While gonadotropins can overcome the effect of these substances in the cumulus oocyte complex, they have no effect on naked oocytes. Cholera enterotoxin, an irreversible activator of adenylate cyclase, maintains developmental arrest in cultured cumulus oocyte complexes but not in naked oocytes. Preparations of luteinizing hormone can partially overcome the effect of cholera enterotoxin in the complexes. Furthermore, the acceleration of oocyte maturation in vitro observed in the presence of gonadotropins, which is seen in cumulus oocyte complexes, can be mimicked by stripping the oocyte of its associated cumulus cells. The results of these and other studies suggest that: (1) the cytoplasmic levels of cyclic AMP in the isolated oocyte are high enough to maintain meiotic arrest if a phosphodiesterase inhibitor is present; (2) the oocyte contains an active phosphodiesterase; (3) the oolemma may lack the adenylate cyclase system and; (4) gonadotropins seem to affect the oocyte indirectly, via the cumulus cells, possibly by interrupting communication between the two cell types.     

Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone

Experiments were performed to determine if elevation of cumulus cell cAMP results in an increase in mouse oocyte cAMP while the heterologous gap junctions were intact. Both follicle-stimulating hormone (FSH) and cholera toxin induced a marked increase (>20-fold) in intracellular cAMP in isolated mouse cumulus cell-oocyte complexes in the presence of 3-isobutyl-1-methyl xanthine (IBMX). Concomitantly, both FSH and cholera toxin transiently inhibited resumption of meiosis of cumulus cell-enclosed but not denuded oocytes. The transient nature of the inhibitory effect produced by either FSH or cholera toxin was correlated with the cAMP level in the cumulus cell-oocyte complex. The inhibitory effect, however, was apparently not due to movement of cumulus cell cAMP to the oocyte via the functional heterologous gap junctions between cumulus cells and the oocyte. Radioimmunoassay of cAMP in oocytes free of attached cumulus cells or cumulus cell-enclosed oocytes exposed to either FSH or cholera toxin revealed that both groups of oocytes contained similar amounts of cAMP (about 0.14 fmole/oocyte). Metabolic labeling of cumulus cell-oocyte complexes with [³H]adenosine followed by incubation with either FSH or cholera toxin resulted in a marked increase in the amount of radiolabeled cAMP compared to that in unstimulated complexes. However, similar amounts of radiolabeled cAMP were found in oocytes derived from either stimulated or unstimulated complexes. Thus, we have not detected, using two methods of assay, that increasing the cAMP content of the cumulus cells results in any increase in the cAMP content of the oocyte. The apparent compartmentalization of cumulus cell cAMP elevated in response to either FSH or cholera toxin was not due to disruption of intercellular communication between the two cell types during the incubation; metabolic cooperativity was present between the two cell types and molecules of similar molecular weight and charge relative to that of cAMP were rapidly equilibrated between the two cell types. Testosterone potentiated the FSH/cholera toxin-induced transient inhibition of maturation of cumulus cell-enclosed oocytes. However, testosterone did not increase cAMP accumulation produced by either FSH or cholera toxin, decrease the rate of cAMP degradation, or promote movement of cumulus cell cAMP to the oocyte. Since cAMP elevated in response to FSH or cholera toxin appeared to be compartmentalized to cumulus cells and since neither FSH, cholera toxin, nor testosterone inhibited resumption of meiosis in denuded oocytes, it appears that the inhibitory effect promoted by FSH or cholera toxin is directly mediated by an agent other than cAMP, although cAMP generation is required for its action and that cumulus cells mediate the inhibition. These results are discussed in terms of a possible role of cAMP and steroids in regulating maturation in the mouse.     

Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin

We have recently reported that the adenylate cyclase activator, forskolin, induces in the rat ovarian follicle both cAMP accumulation and oocyte maturation. We demonstrate here, on the other hand, that the spontaneous maturation in vitro of isolated rat cumulus-enclosed oocytes is inhibited by forskolin. The inhibitory effect of forskolin is dose dependent with an ED50 at 15 microM. Forskolin inhibition decreases gradually with time, being completely relieved by 20 h of culture. Methylisobutylxanthine significantly prolongs the duration of the inhibitory action of forskolin. In addition to its inhibitory effect on oocyte maturation, forskolin triggers the cumulus-oocyte complex to generate cAMP. Cyclic AMP accumulation is maximally stimulated by 100 microM of forskolin with an ED50 at 60 microM. The potency of the cumulus-oocyte complex to respond to forskolin in terms of cAMP accumulation decreases with time. The pattern of the decrease in the potency of the cumulus-oocyte complexes to generate cAMP corresponds with the relief of its inhibitory influence on the oocyte. These results indicate that inhibition of maturation of the cumulus-enclosed oocyte may be coupled to elevation of cAMP levels in the cumulus-oocyte complex. As isolated cumulus-free oocytes are not inhibited by forskolin, we suggest that in the cumulus-enclosed oocyte system, cAMP generated by the cumulus cells is apparently transferred to the oocyte and maintains it in a meiotically arrested state. Maturation in this system occurs upon relief of inhibition which results from cessation of cAMP generation by the cumulus cells.     

cAMP in ovine oocytes: localization of synthesis and its action on protein synthesis, phosphorylation, and meiosis

In the first series of experiments, the source of cAMP in the sheep oocyte was studied. Cholera toxin was shown to be a potent stimulator of cAMP in isolated sheep oocytes, demonstrating the presence of adenyl cyclase. There was no evidence for transmission of cAMP from stimulated myocardial cell monolayers to cumulus-enclosed oocytes even though the existence of a concentration gradient of cAMP and of intercellular communication were demonstrated. However, gonadotrophin-stimulated follicle shells were able to induce a rise in the cAMP content of denuded or cumulus-enclosed oocytes in the same dish, independently of cell contact. Further experiments were designed to study the effects of a cholera toxin-stimulated rise in cAMP on the maturation of oocytes. When applied to cumulus-oocyte complexes, cholera toxin did not block germinal vesicle breakdown (GVBD), nor the accompanying changes in protein synthesis and phosphorylation, although there was evidence for a delaying effect. There were, however, indications that the toxin was inducing abnormalities that became gross when the concentration was raised to 1 microgram/ml. This high concentration of cholera toxin was able to block the maturation of oocytes in intact, gonadotrophin-treated follicles, although once again abnormalities were evident. We conclude that the role of cAMP in the maturation of the sheep oocyte is different from that proposed in the mouse.     

Regulation of murine oocyte meiosis: evidence for a gonadotropin- induced, cAMP-dependent reduction in a maturation inhibitor

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

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.     

Testosterone response in arginine vasopressin desensitized immature rat testis

Direct injection of arginine vasopressin into immature rat testis inhibited basal testosterone synthesis. Simultaneous injection of arginine vasopressin with luteinizing hormone, norepinephrine or cholera toxin inhibited these agonists - induced testosterone response. In arginine vasopressin - desensitized testis, cAMP response to luteinizing hormone, norepinephrine and cholera toxin was not disturbed. However, testosterone response to luteinizing hormone, norepinephrine or cholera toxin was drastically reduced in arginine vasopressin-desensitized testis. This shows that the increased cAMP generated by luteinizing hormone, norepinephrine or cholera toxin in arginine vasopressin desensitized testis did not cause increase in steroidogenesis. This could be due to a lesion in steroidogenic pathway beyond cAMP generation caused by arginine vasopressin.     

Induction and Inhibition of Meiotic Maturation in Follicle-enclosed Mouse Oocytes by Forskolin

A continuous exposure of follicle-enclosed mouse oocytes to ovine luteinizing hormone (LH, 10 μg/ml) in vitro resulted in a 3-fold elevation of CAMP levels in the follicle cells, but not the oocytes, with subsequent oocyte maturation. When follicle-enclosed oocytes were exposed to forskolin (0.01–10 μM) for 2 hr and then incubated in forskolin-free medium (transient exposure group), oocytes underwent germinal vesicle breakdown in a dose-dependent manner. In contrast, a continuous exposure of the follicles to forskolin (10 μM) for up to 10 hr failed to induce resumption of meiosis. Follicle cell cAMP levels increased within 2 hr after the initial exposure to forskolin, and thereafter decreased rapidly regardless of whether forskolin treatment was transient or continuous. A similar transient increase in oocyte cAMP levels was observed after transient or continuous treatment with forskolin. It was evident, however, that at any time examined oocyte cAMP levels were consistently higher in the continuous exposure group than in the transient exposure group. Furthermore, a continuous exposure to forskolin also blocked LH-induced meiotic maturation. These findings suggest that elevated levels of cAMP in the oocyte block meiotic maturation in mouse oocytes. The present results further suggest that an increase in follicle cell cAMP levels is essential to the LH-induced meiotic maturation.     

Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10(-6), 10(-4) and 10(-2) M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10(-2) M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10(-2) M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10(-2) M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.     

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.     

Studies of microbial toxins in Xenopus laevis oocytes

Xenopus laevis oocytes have been incubated or microinjected with cholera and diphtheria holotoxins or their respective isolated fragments A and B. Effects on progesterone-induced maturation, protein synthesis and cAMP levels were observed. Xenopus laevis oocytes were highly susceptible to cholera toxin upon incubation as evidenced by the increase of cAMP (two-fold increase in cAMP with 0.1 nM cholera toxin) and the blockade of progesterone-induced maturation. When isolated cholera toxin fragments A or B were incubated with oocytes, no activity could be detected. However, microinjection of cholera toxin fragment A into oocyte was able to mimic the effects of incubated holotoxin. Microinjection of cholera toxin B fragment was only effective at very high concentrations, probably due to trace contaminations by the A fragment. On the other hand, Xenopus laevis oocytes were very resistant to diphtheria toxin action upon incubation, a result attributable to lack of specific membrane receptors since, after microinjection of diphtheria toxin A fragment into oocytes, inhibition of protein synthesis was demonstrated. By simultaneous microinjection of highly radioactive adenine-labelled NAD and diphtheria toxin fragment A into oocytes, radioactive ADP ribosylation of the elongation factor 2 (EF₂) was observed. It is proposed that Xenopus laevis oocytes provide a new experimental approach for studying the mechanisms of action of microbial toxins.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.     

Inhibition of maturation and metabolism in rat oocytes by cyclic amp.

It is known that dibutyryl cyclic AMP (dbcAMP) and theophylline inhibit the spontaneous maturation of isolated mouse oocytes. The present study demonstrates that dbcAMP (0.01-1.0 mM) as well as cyclic AMP (cAMP, 10 mM) and a phosphodiesterase inhibitor (IBMX, 0.01-1.0 mM) prevent spontaneous maturation of isolated rat oocytes. As reported earlier an increase in oxygen consumption by the oocyte was found following maturation. When the oocytes were cultured in the presence of dbcAMP or cAMP no change in respiration occurred during culture. These results argue against the theory that cAMP acts as a direct mediator of the action of luteinizing hormone (LH) on oocyte maturation. Furthermore they suggest that changes in oocyte energy metabolism are closely related to the maturation process.     

Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non-hormone-treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium-deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation-promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin-like protease plays a role(s) in regulating the oocyte meiotic maturation process.     

Expression of endothelial nitric oxide synthase in the porcine oocyte and its possible function

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitro development. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.     

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells

The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells.     

Discordance in the effects of interleukin-1 on rat granulosa cell differentiation induced by follicle-stimulating hormone or activators of adenylate cyclase

Recombinant human interleukin-1 (IL-1) inhibits the follicle-stimulating hormone (FSH)-induced development of luteinizing hormone (LH) receptors and suppresses progesterone secretion in cultured rat granulosa cells. Since activation of adenylate cyclase by FSH is considered to be the primary second messenger system responsible for differentiation of granulosa cells, we examined whether IL-1 could alter the FSH, cholera toxin, or forskolin-induced accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) from these cells. In addition, we sought to determine if IL-1 could influence differentiation induced by the cAMP analog, 8-bromo cAMP. Cells collected from ovaries of immature, diethylstilbestrol-treated rats were stimulated to differentiate by addition of FSH, cholera toxin, forskolin, or 8-bromo cAMP to the cultures. IL-1 or interleukin-2 (IL-2) was added to some of the tubes, and the primary cultures were incubated for various periods of time. At the end of the culture, the tubes were centrifuged, the medium was saved for progesterone and cAMP radioimmunoassay, and the cells were assayed for specific 125I-human chorionic gonadotropin (hCG) binding to determine the number of LH receptors. In the presence of FSH, IL-1, at a dose as small as 5 ng/ml, but not IL-2, significantly inhibited LH receptor formation and suppressed progesterone secretion in a dose-related manner. IL-1 also significantly suppressed FSH-induced cAMP accumulation after 72 h of incubation but did not appear to do so in a dose-related fashion. In the presence of FSH, IL-1 did not significantly alter the protein content of granulosa cells at the end of culture. During stimulation of granulosa cells with cholera toxin, forskolin, or 8-bromo cAMP, IL-1 significantly reduced LH receptor formation compared to that observed in the absence of IL-1. However, in contrast to IL-1 in the presence of FSH, IL-1 significantly augmented the forskolin-induced secretion of progesterone and accumulation of cAMP after 72 h at subsaturating doses of forskolin. Thus, IL-1 appeared to inhibit forskolin-induced and cholera toxin-induced formation of LH receptors even when cAMP levels were elevated. Similar to forskolin, 8-bromo cAMP-stimulated progesterone secretion was significantly enhanced by IL-1, but LH receptor formation was inhibited. Over a 72-h time course at single doses of FSH or forskolin, IL-1 did not affect cAMP accumulation until 48 h of culture, at which time IL-1 significantly suppressed FSH-induced, but augmented forskolin-induced, accumulation of cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)