Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles

Treatment of isolated amphibian ovarian follicles with frog pituitary homogenate (FPH) increases follicular progesterone levels, which, in turn, initiate oocyte maturation. Recent studies have demonstrated that follicular progesterone production requires concomitant protein synthesis at some stage preceding pregnenolone formation. Experiments were carried out to determine whether cholesterol metabolism plays a role in mediating these biochemical and physiological processes. Aminoglutethimide (AGI, and inhibitor of P450 side-chain cleavage enzyme) inhibited FPH-induced intrafollicular progesterone accumulation and oocyte maturation (or germinal vesicle breakdown, GVBD) in a dose-dependent manner. Follicular progesterone accumulation and GVBD were both stimulated, in the absence of FPH, after addition of 25-OH-cholesterol, but not cholesterol, to the culture medium. Higher levels of progesterone were present in defolliculated oocytes as compared to intact ovarian follicles after incubation with 25-OH-cholesterol. The results indicate that the surface epithelium and theca layer in the follicle wall retard 25-OH-cholesterol access to steroid-producing follicle cells. AGI blocked 25-OH-cholesterol-induced accumulation of progesterone and GVBD in defolliculated oocytes, suggesting that 25-OH-cholesterol does not directly induce GVBD and is metabolized by the follicle cells. The capacity of follicles to accumulate progesterone following preincubation with FPH or 25-OH-cholesterol along with AGI was compared. Intrafollicular levels of progesterone increased after AGI- and 25-OH-cholesterol-treated follicles were washed. In contrast, progesterone levels decreased in follicles pretreated with AGI and FPH after washing. The results indicate that considerable 25-OH-cholesterol, but not endogenous cholesterol (FPH stimulation), remains available for steroidogenesis after removal of AGI. A significant, but incomplete, inhibition of progesterone accumulation occurred when follicles were incubated in the presence of 25-OH-cholesterol and cycloheximide. This partial blockage produced by the protein synthesis inhibitor indicates that some basal protein synthesis is required for progesterone accumulation from exogenous 25-OH-cholesterol. We conclude that intracellular cholesterol stores in the follicle wall are utilized to mediate FPH induction of progesterone accumulation and oocyte maturation in amphibian follicles.     

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.     

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains

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ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity

Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6(+/-)) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6(-/-) mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6(-/-) mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6(-/-) mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.     

Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.