Follicle-stimulating hormone promotes histone H3 phosphorylation on serine-10.

FSH promoted the rapid phosphorylation of the nuclear protein histone H3 in immature rat ovarian granulosa cells under experimental conditions that lead to cellular differentiation and not proliferation. FSH-stimulated histone H3 phosphorylation correlated with cAMP-dependent protein kinase A (PKA) activation and translocation of the PKA catalytic subunit to a nuclear-enriched fraction and was inhibited by the PKA inhibitor H89, and histone H3 phosphorylation was stimulated in cells treated with agents that raise intracellular cAMP levels such as forskolin and 8-bromo-cAMP. FSH-stimulated histone H3 phosphorylation in granulosa cells mapped to ser-10, a site previously identified as the PKA phosphorylation site in various mitotically active cells as the mitosis-specific phosphorylation site. Injection of the FSH analog PMSG to immature rats, which is known to stimulate granulosa cell proliferation as well as differentiation, also promoted histone H3 phosphorylation on ser-10 in granulosa cells. These results establish that FSH-stimulated histone H3 phosphorylation in granulosa cells is linked not only to granulosa cell mitosis but also to granulosa cell differentiation and that FSH-stimulated histone H3 phosphorylation on ser-10 in isolated granulosa cells is mediated by PKA. These results also identify the PKA-dependent histone H3 phosphorylation as an early nuclear protein marker for FSH-stimulated differentiation of granulosa cells. Based on the recently described function of histone H3 as a coactivator of transcription, these results are consistent with the hypothesis that phosphorylated histone H3 may facilitate PKA-dependent gene transcription in granulosa cells leading to the preovulatory phenotype.

Characterization of recombinant RI beta and evaluation of the presence of RI beta protein in rat brain and testicular extracts.

Based upon recent reports that the mRNA from the regulatory (R) RI beta subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RI beta protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RI beta. Recombinant RI beta eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RI alpha. As predicted by its amino acid sequence homology to RI alpha, recombinant RI beta was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3':5'-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RI alpha (47 kDa) and recombinant RI beta (53 kDa). However, recombinant RI beta exhibited an unexpectedly basic pI of 6.65-6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RI beta, 8-N3[32P]cAMP-labeled RI beta was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RI beta. The 53-kDa RI beta was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N3[32P]cAMP-labeled RI beta was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic pI of RI beta allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RI beta mRNA and protein.

Follicle-stimulating hormone regulation of A-kinase anchoring proteins in granulosa cells.

It has been well established that the biochemical and morphological changes during maturation of granulosa cells that are induced by follicle-stimulating hormone (FSH) occur through the elevation of intracellular cAMP and consequent activation of the cAMP-dependent protein kinase (PKA). In this report we show that FSH action alters the expression of A-Kinase Anchoring Proteins (AKAPs), which function to target the subcellular distribution of the type II PKA. Exposure of granulosa cells grown in primary culture with FSH and estradiol for 72 h resulted in the up-regulation of an 80-kDa AKAP and the RII beta subunit of PKA, whereas cells grown in control medium containing only estradiol produced a time-dependent increase of a 140-kDa AKAP. RII overlays performed with [32P]RII alpha preferentially detected RII-binding bands of 80 and 95 kDa compared to blots probed with [32P]RII beta, suggesting that FSH may alter the subcellular location of PKA in an isoform-specific manner. FSH treatment causes a translocation of RII alpha from the particulate to the cytosolic fraction coincident with the induction of the 80-kDa AKAP, which is also predominately cytosolic. These data suggest that FSH promotes a redistribution of the type II PKA holoenzyme through the selective induction of an RII isoform-specific AKAP.

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.

Hormonal regulation of the type III isoform of C-kinase in porcine ovarian tissues.

C-kinase activity is notably increased in corpora lutea (CL) compared to preovulatory follicles of porcine ovaries. Our purpose was to identify the C-kinase isoform(s) involved in this increase and to examine the expression of C-kinase in ovarian tissues at different stages. The major component of C-kinase activity in the CL was isoform III, with a molecular weight (M(r)) of 80,000. Minor activities were attributed to the type II isoform (M(r) = 80,000) and an unidentified C-kinase activity (M(r) = 77,000). C-kinase was not partitioned differently in preovulatory follicles and CL as demonstrated by tissue homogenization in the presence of a detergent and increased chelators. A 3-fold increase in immunoreactive C-kinase was detected in postovulatory follicles relative to preovulatory. A second, nearly 3-fold increase in C-kinase was detected in mature CL relative to postovulatory follicles. These increases are examined in the context of the complex hormonal regulation of the porcine CL.

cAMP-dependent protein kinase isozymes in porcine follicles and corpora lutea.

Our purpose was to identify regulatory (R) subunits and their associations with catalytic (C) subunits to form cAMP-dependent protein kinase (A-kinase) holoenzymes in select porcine ovarian tissues during follicular differentiation. Soluble extracts of small and preovulatory follicles and corpora lutea (CL) were separated on DEAE-cellulose chromatography. R subunits were labeled with 8-N3[32P]cAMP or with [gamma-32P]ATP under RII autophosphorylation conditions and were identified by molecular weight (Mr) determination on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as cross-reactivity of unlabeled subunits with anti-R antibodies. A-kinase holoenzymes and C subunit-free R (free R) subunits were distinguished on the basis of DEAE elution position and sedimentation position on sucrose density gradient centrifugation of phosphotransferase and [3H]cAMP binding activities. In small and preovulatory follicles and CL we identified a minor peak of type I A-kinase containing RI alpha (Mr = 47,000) and a major peak of type II A-kinase holoenzyme containing two RII isoforms (Mr = 52,000 and 56,000). Notable amounts of free RI alpha eluted between the type I and II holoenzymes in all three tissues. Neither of the holoenzymes nor free RI alpha was regulated as a function of follicular differentiation or CL formation. In contrast, free RII subunits were moderately increased in preovulatory follicles relative to levels in small follicles and CL. We conclude that only the RII subunits are hormonally regulated in developing follicles, and in tissues which express both RI and RII subunits, the RII subunits preferentially associate with C subunits to form the dominant holoenzyme despite the presence of significant amounts of RI.

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.

Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin.

The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.

Steroid-induced final maturation in brook trout (Salvelinus fontinalis) oocytes in vitro: the effects of forskolin and phosphodiesterase inhibitors.

The effects of phosphodiesterase inhibitors and forskolin on steroid-induced germinal vesicle breakdown (GVBD) were investigated in brook trout (Salvelinus fontinalis) oocytes using an in vitro incubation technique. Follicles were first treated with a collagenase solution to remove the follicle wall. Denuded oocytes were examined, using scanning electron microscopy. In all experiments GVBD was induced by the use of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one. Cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured (by protein-binding assay) in control and forskolin-treated oocytes. Collagenase treatment removed a majority of the follicle wall, as shown by scanning electron microscopy. Partially denuded (PD) oocytes were slightly more sensitive to steroid treatment than intact follicles (IF), as shown by ED50 values; but PD oocytes did not respond to gonadotropin (GTH) stimulation. Both 3-isobutyl-1-methyl-xanthine (IBMX) and SQ20,006 (Squibb) blocked GVBD, but IBMX was more inhibitory. Forskolin also blocked steroid-induced GVBD. Kinetics of inhibition studies were performed using IBMX, forskolin, and cycloheximide. IBMX and cycloheximide inhibited GVBD if added during the first 18 h following steroid stimulation, whereas forskolin blocked GVBD if added within 12 h after steroid treatment. Forskolin, at levels that block GVBD in vitro, significantly increased cAMP in both IF and PD oocytes, but the response of IF was greater than that of PD oocytes.

The effects of forskolin, cAMP, and cyanoketone on steroid-induced meiotic maturation of yellow perch (Perca flavescens) oocytes in vitro.

Intact yellow perch (Perca flavescens) follicles stimulated by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-PG) to undergo germinal vesicle breakdown (GVBD) in vitro were incubated with several agents which have been shown to increase cellular cAMP levels. Two phosphodiesterase inhibitors, SQ20,006 and isobutyl-methyl-xanthine, blocked GVBD at 1.0 mM. At lower levels (0.5, 0.1 mM) there was a dose-response effect and SQ20,006 was more inhibitory. Forskolin at 1.0-20.0 microM blocked steroid-induced GVBD, but levels of 0.1 microM or less were noninhibitory. In time-course experiments, significant inhibition of GVBD was observed when SQ20,006 (1.0 mM) was added within 6 hr after steroid stimulation or forskolin (10.0 microM) was added within 12 hr. When SQ20,006 was administered in 6-hr pulses and then removed, inhibition was observed only when the steroid was given as a 1-hr prepulse which was removed at the start of the incubation period. In this case, GVBD was blocked if the SQ20,006 pulse was given before 18 hr. At 10.0 mM, cAMP completely inhibited GVBD but was noninhibitory at lower levels. However, lower levels of cAMP (1.0, 0.5 mM) and forskolin (0.1 microM) were inhibitory if the follicles were also incubated with 1.0 microgram/ml of cyanoketone, an inhibitor of steroidogenesis. These results indicate that in vitro, increases in cAMP are inhibitory to steroid-induced meiotic maturation but may stimulate steroidogenesis in the follicle wall as well. Furthermore, in vitro steroid-stimulated maturation can be inhibited by increased cAMP for a relatively long time, following steroid treatment.