Hormonal Regulation of Nuclear Cyclic AMP-dependent Protein Kinase Subunit Levels in Rat Ovaries*

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear CAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity label- ing of nuclear extracts with 8-a~ido-[~~P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estro-gen-primed hypophysectomized rats. After follicle- stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, M, = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity label- ing. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity.

could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity.
Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density.
These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.
Many of the actions of gonadotropic hormones in the ovary are mediated by cAMP through CAMP-dependent protein kinases (Marsh, 1975). Cyclic AMP-dependent protein kinase isozymes, through phosphorylation of specific regulatory cellular proteins, are believed to initiate a "built-in" program for action established during early differentiation and development. In the rat ovary, CAMP-binding and CAMP-dependent protein kinase activities increase progressively during post-* This work was supported in part by National Institutes of Health Grant HD12046 and by the Research and Education Fund, Northwestern University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed Dept. of Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. natal development (Lamprecht et al., 1973;DeAngelo et al., 1975) and in response to gonadotropins administered to neonatal rats or adult hypophysectomized rats (DeAngelo et al., 1975). Analyses of changes of protein kinase isozymes have shown that ovarian cytosol of 6-day-old neonates contains largely type I CAMP-dependent protein kinase which changes to a predominantly type I1 protein kinase pattern in 34-dayold and older rats (Jungmann and Hunzicker-Dunn, 1978;Hunzicker-Dunn et al., 1984). The type I1 isozyme similarly increases in rat ovary nuclei as a function of postnatal development (Jungmann and Hunzicker-Dunn, 1978;Hunzicker-Dunn, 1982) but little is known about the hormonal regulation of ovarian nuclear protein kinase levels. Several experiments have suggested that ovarian nuclear CAMP-dependent protein kinase activity is acquired through a CAMP-mediated translocation of cytoplasmic protein kinase subunits (Jungmann et al., 1974;Spielvogel et al., 1977).
In the rat granulosa cell, the level of the cytosolic regulatory subunit RII increases as preantral follicles differentiate into antral follicles (Ratoosh and Richards, 1985;Richards and Rolfes, 1980;Richards et al., 1983, 198:;Darbon et al., 1984). Concomitant with the changes in RII levels, FSH' and cAMP change the CAMP-dependent protein kinase substrate pntterns (Richards et al., 1983;Halpren-Ruder et al., 1980). Since at least some of these CAMP-mediated events can be expected to involve a nuclear action of cAMP and CAMP-dependent protein kinase (Jungmann and Hunzicker-Dunn, 1978), the present studies were undertaken to identify protein kinase subunits RI, RII, and C in rat granulosa cell nuclei and to identify if the nuclear subunits levels are qualitatively and quantitatively modulated by FSH. The coordinated regulation of the nuclear subunits would suggest a regulatory action for the subunits at the nuclear level conceivably participating in regulating the genomic expression of FSH-and CAMP-inducible granulosa cell proteins. gold particles (GAR 20) was purchased from Janssen Pharmaceuticals, Beerse, Belgium.
Animals-Immature female rats (Charles River, Sprague-Dawley), hypophysectomized at the age of 21 days, were purchased from the Hormone Assay Laboratories (Chicago, IL). Seven days after surgery, hormone treatments were initiated as follows: Rats were injected subcutaneously once daily for 4 days with 1.5 mg of 17P-estradiol dissolved in 0.2 ml of propylene glycol and then twice daily for 2 days with 7.5 pg of ovine FSH (in 0.1 ml of phosphate-buffered saline) or only with 0.1 ml of saline. Rats were killed by cervical dislocation 12 h after the last injection.
Preparation of Nuclear Nonhistone Protein Extracts-Nuclear nonhistone protein extracts were prepared as described by us (Maizels and Jungmann, 1983) with slight modifications as follows: Ovaries were homogenized in nuclear isolation buffer (10 mM Tris, pH 7.4, 4 mM MgC12, 50 mM benzamidine, 0.5 mM PMSF, and 1.8 M sucrose). The homogenate was layered onto a 2-ml cushion of nuclear isolation buffer and centrifuged at 105,000 X g for 60 min. The nuclear pellet was collected and nuclear protein was extracted for 30 min with nuclear extraction buffer (10 mM Tris, pH 7.4, 50 mM benzamidine, 0.5 mM PMSF, and 0.35 M NaCl). Insoluble nucleohistone was removed by centrifugation at 105,000 X g for 10 min.
Preparation of Cytosol-Ovaries were homogenized in 10 mM Tris, pH 7.4, 4 mM MgC12, 50 mM benzamidine, 0.5 M PMSF in a ground glass homogenizer. The homogenate was centrifuged for 10 min at 10,000 X g. The resulting supernatant fraction was centrifuged at 105,000 X g for 60 min, yielding a soluble fraction defined as cytosol.
Assay of CAMP-binding and Protein Kinase Activities-Binding of [3H]cAMP to nuclear protein was determined by the Millipore filtration assay according to Rannels et al. (1983). Cyclic AMP-dependent protein kinase activity was assayed as described by us previously (Laks et al., 1981) using the synthetic phosphate acceptor Kemptide as substrate.
Purification of CAMP-dependent Protein Kinase Subunits and Preparation of Antisera-The catalytic subunit was purified from bovine heart as described previously (Schwoch et al., 1980). The regulatory subunits RI and RII were purified to homogeneity from rat liver according to Dills et al. (1979) with the following modifications: Rabbit anti-rat RI or anti-rat RII-protein A-Sepharose 4B affinity columns were used to remove contaminating RI or RII from purified regulatory subunit preparations, respectively.
Preparation of the antisera against bovine heart catalytic subunit and rat liver regulatory subunits RI and RII in rabbits and their characterization have been described in detail (Kuettel et al., 1985).
Miscellaneous Analytical Procedures-DNA concentration was determined according to Burton (1956). Protein was estimated by the method of Bradford (1976). Lactate dehydrogenase activity was assayed spectrophotometrically by converting pyruvate to lactate and measuring the decrease in absorbance of NADH at 340 nm (Derda et al., 1980). Immunocolloidal Gold Electron Microscopy-An immunocolloidal gold electron microscopy procedure was used to localize catalytic and regulatory subunit antigens at nuclear ultrastructural sites and to quantitate antigen staining density. The method was recently described by us in detail (Kuettel et al., 1985).

Criteria for the Isolation and Purity of Nuclei and Establishment of Optimal Conditions for Extraction of Nuclear CAMPdependent Protein Kinase Subunits-In
initial studies we defined the optimal experimental conditions for the isolation of pure nuclei and quantitative recovery of nuclear CAMPdependent protein kinase subunits in ovarian nuclear NaCl extracts. To that purpose we have used a previously developed nuclear isolation procedure which involves homogenization of tissue in buffer containing high molarity sucrose (Laks et al., 1981). Under these conditions, loss of nuclear protein is minimized (Yu, 1975), and the isolated nuclei are not significantly contaminated with non-nuclear material as judged by microscopic examination or biochemical cytoplasmic marker enzyme assay (Laks et al., 1981). When this procedure was used for the isolation of nuclei from rat ovaries, the specific activity of cAMP binding as well as the total nuclear CAMPbinding activity was significantly higher in nuclei isolated in hypertonic sucrose (1.8 M sucrose) buffer than in nuclei isolated in 0.32 M sucrose buffer (Maizels, 1983). With this method, the recovery of nuclei from the ovarian homogenate in 1.8 M sucrose buffer as determined from the DNA concentrations was between 55 and 60%. Evaluation of nuclear morphology and contamination of nuclei by secretory granules and cytosolic particles as well as measurement of marker enzyme activity (Laks et al., 1981) were indicative of "pure" nuclear preparations. The protein:DNA ratio of all nuclear preparations was between 2.3 and 2.0, typical of highly purified nuclei (Tata, 1974).
It was also important for our studies to ascertain that (a) nuclear protein kinase subunits were quantitatively recovered by this isolation procedure and (b) to assure that the subunits were of nuclear origin and not due to contaminations by dissociated cytosolic subunits or undissociated cytosolic holoenzyme. This is particularly critical, since the dissociated cytosolic catalytic subunit will artifactually adhere t o nuclei during their isolation (Keely et al., 1975). In contrast, the dissociated cytosolic regulatory subunits RI and RII do not exhibit artifactual binding activity to nuclei.
To prevent artifactual nuclear binding of the cytosolic catalytic subunit, Keely and co-workers (1975) have developed a procedure, consisting of isolation of nuclei in buffers containing isotonic concentrations of NaCl or KC1, which prevents artifactual binding of cytosolic catalytic subunit to nuclei. Accordingly, we have isolated nuclei in 1.8 M sucrose buffer containing 0.15 M NaC1. On the other hand, since cytosolic RI and RII do not artifactually adhere to nuclei and since inclusion of 0.15 M NaCl in the nuclear isolation buffer led to a considerable loss of nuclear RI and RII (Maizels, 1983), NaCl was deleted from the nuclear isolation buffer when RI and RII were determined.
To ascertain, however, that artifactual nuclear binding of dissociated cytosolic RI and RII as well as of the undissociated holoenzyme did, indeed, not occur, the following experiments were carried out. Cytosol was UV-irradiated in the presence of the photoaffinity label 8-azid0-[~~P]cAMP without and with added nonradioactive cAMP (100 p~) .
The presence of nonradioactive cAMP prevents specific labeling of RI and RII but it does not prevent potential nonspecific labeling of cytosolic proteins other than RI and RII.
After removal of excess free 8 -a~i d o -[~~P ] c A M P , a n aliquot of radioactively labeled cytosol protein (about 25,000 cpm of 32P) was added to an equivalent amount of rat ovarian homogenate. Following isolation of nuclei in buffer containing 1.8 M sucrose but no NaCl, nuclei were extracted with 0.35 M NaCl and 32P radioactivity of the extracts was determined. In each case, less than 1% of the total amount of radioactivity added to the homogenate was recovered in the 0.35 M NaCl extracts (data not shown). There was no difference in the amount of radioactivity recovered regardless of whether the added cytosol had been photoaffinity-labeled in the presence or absence of non-radioactive cAMP indicating that the radioactivity recovered in the nuclear 0.35 M NaCl extracts was due to nonspecifically labeled cytosolic proteins and not to RI or RII. Furthermore, it is shown below (see Figs. 2 and 3) that cytosol but not nuclei contained the RII-54 electrophoretic variant of RII. From this selectivity of RII-54 compartmentation it can be concluded that artifactual binding of cytosolic regulatory subunits to nuclei does not occur. We have already reported (Jungmann et al., 1974) that the protein kinase holoenzymes exhibit little or no binding affinity for calf ovary chromatin. After incubation of nuclei with varying amounts of either the type I or type I1 DEAE cellulose-purified holoenzymes and isolation of nuclei in 1.8 M sucrose buffer, catalytic subunit, RI, and RII concentrations in nuclear extracts were not significantly altered as compared to nuclei that had not been incubated with the holoenzymes. This indicates that contamination of nuclei with cytoplasmic holoenzyme was not a significant problem.
In order to evaluate the quantitative recovery of subunits from the nuclei of unstimulated and FSH-stimulated ovaries, nuclei were extracted successively with buffers containing increasing amounts of NaCl (see Table I). A concentration of 0.35 M NaCl was sufficient to solubilize about 95% of the total nuclear CAMP-binding and catalytic subunit activities. Subsequent extractions with buffers containing higher concentrations of NaCl did not solubilize appreciably more CAMP-binding and catalytic subunit activities. Therefore, in all subsequent experiments nuclear subunits were extracted with buffer containing 0.35 M NaC1.
As the data of Table I show, the recovery of catalytic subunit and CAMP-binding activities in nuclear NaCl extracts from both unstimulated as well as stimulated ovaries was quantitative and of similar efficiency. This is important to note, since it could be argued that the increased levels of subunits in nuclear extracts was due to a lower binding affinity of the subunits to nuclei after FSH treatment resulting in a higher quantitative recovery of the subunits. The data of Table I illustrate that this was not the case.

Effect of FSH on Ovarian Nuclear Catalytic and Regulatory
Subunit Levels-The data of Table I1 show that the specific activities of the subunits were lower in nuclear extracts as compared to cytosol. FSH stimulation led to a 2-3-fold increase of both the specific CAMP-binding and catalytic subunit activities in nuclei. In the cytosol, on the other hand, FSH stimulation led only to an increase of the specific CAMPbinding but not of the catalytic subunit activity in agreement with previously published data (Richards and Rolfes, 1980;Darbon et al., 1984). Taking the protein concentration in nuclei and homogenate and a recovery of nuclei of 55% into consideration, it was calculated that about 2.7% of the total cellular catalytic subunit activity was present in nuclei from unstimulated ovaries. FSH stimulation increased this percentage to 4.7%. Similarly, FSH increased the nuclear CAMPbinding activity from 4.5% to 11.9% of the total cellular activity.
A more detailed study of the changes of nuclear catalytic subunit levels is illustrated in Fig. 1. FSH treatment of estrogen-primed hypophysectomized rats for 1 or 12 h, 1 and 2 days, increased catalytic subunit activity of nuclear extracts as compared to untreated controls (Fig. 1, panels A and B ) .
Maximal increase of kinase activity was observed after 1 day of FSH treatment (see Fig. 1, panel B). Phosphorylation of endogenous nuclear substrates was relatively low (Fig. 1, panel  A ) and did not contribute significantly to the level of phosphorylation seen with Kemptide as substrate. The priming of hypopysectomized rats with 170-estradiol without subsequent FSH treatment did not affect nuclear catalytic subunit activity. Saturating concentrations of heat-stable protein kinase inhibitor led to an approximate 50% inhibition of the total nuclear protein kinase activity in unstimulated ovaries and approximately 75% inhibition of the total kinase activity in FSH-stimulated ovaries (data not shown). This indicates that the FSH-mediated increase of nuclear protein kinase activity was primarily due to an increase of catalytic subunit activity and not to a modulation of other nuclear CAMP-independent protein kinase activities.
Estimation of the nuclear regulatory subunit levels by the [3H]cAMP-binding assay allows measurement of the total sum of RI and RII, but this method does not discriminate between the two subunits. Since FSH markedly increased nuclear [3H]cAMP binding activity (see Table 11), we evaluated the effect of FSH on the patterns of nuclear regulatory subunits by photoaffinity labeling with 8-a~ido-[~~P]cAMP. For comparative purposes, cytosolic RI and RII were also photoaffinity labeled and identified. After electrophoresis and autoradiography several types of photoaffinity-labeled polypeptides were reproducibly observed (Fig. 2). The regulatory subunit RI is represented by a single band of an apparent molecular weight M, = 49,000 (Rubin and Rosen, 1975;Nimmo and Cohen, 1977;Krebs and Beavo, 1979;Flockhart and Corbin, 1982;Schwartz andRubin, 1983, 1985). The electrophoretic patterns of RII, on the other hand, are heterogeneous and differ in nuclear extracts and cytosol. The nuclear RII subunit appears to consist of a 52,000-dalton

TABLE I
Concentration of CAMP-binding and catalytic subunit activities in NaCl extracts of rat ovary nuclei Hypophysectomized rats were primed with 17&estradiol and subsequently injected with FSH for 2 days as described in "Experimental Procedures." Nuclei were isolated in nuclear isolation buffer without (to determine CAMP-binding activity) or with 0.15 M NaCl (to determine catalytic subunit activity). Nuclear extracts were prepared by successive extractions of nuclei with buffer containing 0.35,0.6, and finally 1.0 M NaC1.

TABLE I1
Cyclic AMP-binding and catalytic subunit activities in nuclei and cytosol of rat ovaries stimulated with FSH Hypophysectomized rats were hormonally treated, and ovarian nuclei were isolated as described in the legend to Table I. Cytosol (105,000 X g supernatant fraction) was prepared as described under "Experimental Procedures." Nuclear extracts were prepared with extraction buffer containing 0.35 M NaCI. Assays were carried out as described in Table I peptide (RII-52) in unstimulated ovaries. In FSH-stimulated ovaries, two variant forms of RII are seen in nuclei, e.g. RII-52 as well as a 51,000-dalton peptide (RII-51) (see Fig. 2). The efficiency of photoaffinity labeling of both forms increased markedly after FSH stimulation suggesting an effect of FSH on nuclear RII-51 and -52 levels. However, because of inefficiency and variability of ligand exchange, radioactive labeling, autoradiographic exposure times, etc., photoaffinity labeling is not a suitable method to obtain precise quantitation of the regulatory subunits and immunochemical methods were employed for their precise determination (see below). The cytosolic 54,000-dalton RII variant (RII-54) was not identified in nuclear extracts. The reason for this could be an inefficient photoaffinity labeling of nuclear RII-54, the complete absence of RII-54 from nuclei, or the loss of small amounts of RII-54 during nuclear isolation.
To determine if the variant forms of RII could also be distinguished on the basis of charge, regulatory subunits were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis after photoaffinity labeling. For comparative purposes, the two-dimensional electrophoretic pattern of cytosolic regulatory subunits from FSH-treated ovaries is shown in Fig. 3, panel C. As previously reported (Jahnsen et al., 1986), several regulatory subunit variants can be identified in the cytosol. Although we were not able to achieve a better resolution, several other isoelectric RII variants, identified as RII-51, RII-51.5, and RII-52, are visible on the autoradiograph in addition to the RII-54 variant. In ovarian nuclear extracts from FSH-treated rats, RII was also resolved into several isoelectric variants (Fig. 3, panel A ) conceivably identical with the cytosolic RII-51, -52, and -51.5 forms. As on the onedimensional gel (Fig. 2), the RII-54 variant could not be identified in the nuclear extracts. The autoradiograph density pattern of nuclear RII from untreated rat ovaries is considerably less dense particularly at the more basic PI values (Fig.   3, panel B). However, the lack of resolution of the spots does not allow a precise identification of the variants. Comparison of the relative autoradiograph densities in panels A and B of Fig. 3 confirms the FSH-mediated increase of photolabeling of RII-51, -52 seen in Fig. 2. The distribution of labeled spots on the two-dimensional gels (Fig. 3,panels A, B, and C) shows that the RI subunit exhibits charge heterogeneity, since three isoelectric variants can be identified. The RI variants have previously been observed after two-dimensional gel analysis of rat ovary cytosol (Jahnsen et al., 1986). The photoaffinity labeling of RI and RII seen in Fig. 3, panels A , B, and C, was specific for CAMP-binding sites, since photoaffinity labeling of RI and RII was prevented by the addition of excess competing cAMP to the reaction mixtures (Fig. 3, panel D).

Quantitation of Protein Kinase Subunits in Nuclear Nonhistone Protein Extracts by Enzyme-linked Immunosorbent
Assay-An enzyme-linked immunosorbent assay was used to quantitate FSH-mediated changes of nuclear protein kinase subunit levels. The competition curves obtained in a representative ELISA experiment are shown in Fig. 4. For the RII and C subunits, significantly less nuclear protein from FSH- (0 subunits of CAMP-dependent protein kinase in ovarian nuclear extracts from estrogen-primed unstimulated (0) and estrogen-primed hypophysectomized rats stimulated with FSH for 2 days (0). A/Ao = absorbance measured in the presence of competing antigen relative to that measured in the absence of competing antigen (i.e. 100% absorption). stimulated ovaries was needed to achieve the same degree of inhibition of antibody-antigen binding seen with nuclear protein from unstimulated ovaries. A similar ELISA experiment showed no difference of nuclear RI subunit levels in stimulated and unstimulated ovaries. These experiments indicate a selective increase of RII and C after FSH stimulation but not of RI. Table I11 summarizes the ELISA data from several

TABLE 111
Quantitation of nwlear CAMP-dependent protein kinase subunits in rat ovaries by enzyme-linked immunosorbent assay Hypophysectomized rats were primed with 170-estradiol and subsequently injected with FSH for 2 days as described under "Experimental Procedures." Nuclear non-histone protein was isolated and analyzed for protein kinase subunits by ELISA as described in Fig.  4. Absolute values were calculated by comparison of the competition curves shown in Fig. 4 with the parallel curves obtained with standard highly purified preparations of rat liver RI, RII, and C. Results reported are the mean f S.E. of three experiments. independent experiments. We observed an approximate 2fold increase of the C and RII content in ovarian nuclei from FSH-treated rats as compared to untreated rats.

Effect of FSH on Protein Kinase Subunit Density in Granulosa and Thecal Cells Determined by Immunogold Electron
Microscopy-We have recently developed a post-embedding immunocolloidal gold electron microscopy technique which allows the ultrastructural localization and morphometric It has been shown (Keely et al., 1975) that due to its charge properties the catalytic subunit, but not the regulatory subunits, will bind artifactually to nuclei after cell disruption. When measuring nuclear catalytic subunit activity, it becomes necessary, therefore, to isolate nuclei under conditions, e.g. inclusion of 0.15 M NaCl or KC1 in the isolation buffer, which prevent the nonspecific nuclear binding of the catalytic subunit. In contrast, since the regulatory subunits do not exhibit the artifactual binding behavior, it is not necessary to include isotonic NaCl or KC1 in the nuclear isolation buffer when measuring nuclear regulatory subunit levels. In fact, we and other investigators have shown that the presence of 0.15 M NaCl in the isolation buffer leads to a marked loss of nuclear regulatory subunits and other nuclear nonhistone proteins (Maizels, 1983;Comings and Tack, 1973).* It became necessary, therefore, to isolate nuclei by two slightly differing Nu Cy Nu Cy procedures (isolation buffer with and without 0.15 M NaCl) FIG. 5. Morphometric quantitation of CAMP-dependent depending on which subunits were determined. Additionally, protein kinase subunit localization in rat ovarian granulosa through the use of high molarity sucrose in the nuclear and thecal cells. Ovarian tissue thin sections were prepared from isolation buffers, we have optimized the isolation procedure estrogen-primed unstimulated (open bars) and estrogen-primed FSHin such a way that a loss of inherent nuclear subunits was stimulated (2 days) (striped bars) hypophysectomized rats. Fixation minimized.
of tissue was carried out with 2.5% glutaraldehyde, and embedding was in low viscosity resin. Mounted tissue sections were incubated

The
Of this study demonstrate that the with the respective antiserum or preimmune serum followed by in-dependent Protein kinase subunits Rh RII-519 -529 and c are cubation with goat anti-rabbit immunoglobulin linked to 20-nm col-present in rat ovary nuclei and that the nuclear levels of RIIloidal gold particles. Tissue sections were examined using a JEOL-51, -52, and C, but not of RI and RII-54, are regulated by JEM-100 cx electron microScoPe and PhotoPaPhed. Morphometric FSH in estrogen-primed hypophysectomized rats. These data quantitation of immunogold staining was carried out in at least 10 were experimentally obtained through measurements of the different cells from each of four different immunogold preparations.
of colloidal gold particles per cellular component area (gold particles/ through qualitative evaluation Of subunits pm2 k S.E.). For a detailed description of the procedure see Kuettel by photoaffinity labeling, and through quantitation of nuclear et al. (1985). Panels A, B, and C, nuclear (Nu) and cytoplasmic (CY) subunits by two different immunochemical methods. Whereas staining density of granulosa cells; panels D, E, and F, nuclear and the immunochemica~ do not distinguish between cytoplasmic staining density of thecal cells. Dotted bars, thin sections nuclear RII variants, analysis of photoaffinity~~abe~ed regutreated with preimmune serum. latory subunits by one-and two-dimensional gel electropho-Immunogold labeling (staining density) is expressed as the number cAMP-binding and subunit activities, quantitation of the relative density of nuclear antigen in fixed thin tissue sections (Kuettel et al., 1985;Jungmann et al., 1988). Application of this method for the analysis of nuclear RI, RII, and C in the ovaries of normal 33-day-old rats showed the association of all three subunits with subnuclear structures of granulosa as well as of thecal cells (Kuettel et al., 1985).
We have used this technique to evaluate and compare nuclear and cytoplasmic subunit density in thecal and granulosa cells from unstimulated and FSH-stimulated estrogenprimed hypophysectomized rats (see Fig. 5). FSH caused an approximate 1.8-fold increase of RII antigen density in nuclear as well as cytoplasmic areas of granulosa cells (Fig. 5,  panel A ) . In contrast, theca cell RII density was not altered in either nuclear or cytoplasmic areas (panel D). The catalytic subunit density was increased only in the nuclear but not in cytoplasmic areas of granulosa and thecal cells (panels B and E). FSH had not effect on RI subunit density in either granulosa (panel C ) or thecal cells (panel F).

DISCUSSION
Through the application of complementary immunochemical, immunocytochemical, and biochemical techniques, we have characterized the types and hormonal control of the subunits of CAMP-dependent protein kinase in nuclei of differentiating rat ovaries. Since our studies focused on subunits specifically localized in nuclear substructures, it was critical and essential to develop a nuclear isolation procedure in which we carefully controlled not only artifactual adherence of cytoplasmic subunits to nuclei but, just as importantly, loss of subunits inherently localized in the nucleus. resisidentified atleast two RII variants in nuclei, RII-5iand -52, and possibly a third variant exhibiting a slightly more basic isoelectric point than RII-51 and -52.
The unique feature of the RII-51, -52 variants is that their nuclear level is hormonally regulated, whereas that of RII-54 is not. In fact, we were not able through photoaffinity labeling to identify the cytosolic RII-54 variant in nuclei. This finding is of particular interest because it has previously been demonstrated that only ovarian cytosolic RII-51 and -52, but not RII-54, are hormonally regulated in estrogen/FSH-treated hypophysectomized rats . Thus, the presence of RII-51 and -52 in nuclei and their hormonal regulation suggests a selective nuclear regulatory action for these subunits. Based on peptide mapping analysis and cDNA structure analysis, Jahnsen et al., (1985Jahnsen et al., ( , 1986 have demonstrated a structural similarity between RII-51 and RII-52 and have suggested that RII-52 may be a post-translational modification of RII-51. RII-54, on the other hand, is structurally distinct from RII-51. It should be noted that the FSH-mediated increase of nuclear and cytosolic specific subunit activities was markedly higher in nuclei than in cytosol (see Table 11). Furthermore, the catalytic subunit activity increased only in nuclei but not in cytosol. This indicates that the nuclear changes did not merely mirror any changes of subunit activity in the cytoplasm and argues against a change of nuclear subunit levels due to contamination of nuclei by a small, constant fraction of cytoplasmic subunits. It also suggests a selective increase of subunits in nuclei, a process which is regulated separately from the modulation of cytosolic subunits. * E. T. Maize1 and R. A. Jungmann, unpublished observations. The immunocolloidal gold electron microscopy method provided us with a valuable complementary analytic approach for the study of nuclear subunits. The method circumvents cell rupture and the ensuing potential for either artifactual loss of nuclear components or binding of cytosolic proteins to nuclei during nuclear isolation. Furthermore, it allowed a semi-quantitative evaluation of protein kinase subunits and a selective analysis of granulosa as well as thecal cells. In general agreement with the ELISA and biochemical data, the immunocolloidal gold method indicated that RI, RII, and C were localized in the nuclear and cytoplasmic areas of granulosa and thecal cells (Fig. 5). RI staining density was not altered by FSH. In contrast, FSH treatment resulted in a selective, cell-specific effect on nuclear RII and C levels. While FSH increased the RII density in the nuclear and cytoplasmic areas of granulosa cells (Fig. 5, panel A ) , RII density was unchanged in thecal cells (Fig. 5, panel D). On the other hand, catalytic subunit density was increased only in the nuclear but not cytoplasmic areas of granulosa and thecal cells (Fig.  5, panels B and E ) . The reason why FSH action requires modulation of nuclear RII-51, -52, and C in granulosa cells but only of nuclear C in thecal cells remains to be elucidated. Under the experimental conditions the protein kinase subunits are relatively resistant to the destructive potential of fixation, dehydration, and embedding. However, direct quantitative comparisons between nuclear and cytoplasmic subunit density should only be done with caution because possible differences of the binding affinity of the subunits to nuclear substructures and extranuclear regions, and a possible preferential loss of cytoplasmic antigen during tissue processing, may make these comparisons imprecise. Additionally, because of differing antibody-antigen affinities, a quantitative comparison of the labeling densities of the various subunits is not feasible.
While it has previously been shown that the FSH-mediated increase of cytosolic RII is due to increased RII protein synthesis and elevated RII mRNA levels (Richards and Rolfes, 1980;Richards et al., 1983Richards et al., , 1984Darbon et al., 1984;Ratoosh and Richards, 1985;Jahnsen et al., 1986;Ratoosh et al., 1987;Hedin et al., 1987), it is of interest to consider the molecular mechanism of modulation of nuclear RII and C. Since granulosa cells proliferate rapidly during follicular development and gonadotropin stimulation (Hirshfield, 1985), it is possible that nuclei acquire elevated levels of CAMPdependent protein kinase in the form of its undissociated holoenzyme or as dissociated subunits at the time of meiosis and chromatin assembly. However, because the addition of cAMP at various concentrations to ovarian nuclei fails to stimulate protein kinase activity (Jungmann and Kranias, 1977), we consider the presence of undissociated holoenzyme in nuclei unlikely. Also, the increase of nuclear RII levels after hormonal stimulation can not be explained by the presence and activation of nuclear holoenzyme. Based on an evaluation of all experimental data, elevation of nuclear subunit levels is best explained by either a FSH-mediated packaging of dissociated subunits into chromatin at the time of meiosis or, alternatively, as a consequence of translocation of subunits from the extranuclear space into the nucleus. This conclusion is strongly supported by findings that quantitation of nuclear subunit levels by two independent immunochemical methods, using antisera which interact efficiently with both the dissociated as well as undissociated subunits in the holoenzyme form,3 demonstrates a net increase of immunoreactive nuclear RII and C antigen. artifactual association of subunits to nuclear structures during cell homogenization, we have carefully assessed and controlled the nuclear isolation procedure to avoid such artifacts. In fact, our experimental findings do not support the notion of a nonspecific modulation of nuclear subunits. The selective nuclear increase of C and RII-51 and -52 but not of RI or RII-54, and the general agreement between the biochemical, ELISA, and immunocytochemical data argue strongly for a specific modulation and against a nonspecific binding and translocation of subunits. The previous findings that different forms of RII accumulate in estrogen/FSH-stimulated ovaries (Richards and Rolfes, 1980;Jahnsen et al., 1985;1986) and our demonstration of a modulation of nuclear levels of RII-51, -52, and C suggest that certain variant forms of RII as well as C are selectively required in the nucleus during ovarian differentiation. While the exact biological roles of the nuclear subunits remain to be determined, the degree of selective nuclear localization and the magnitude of modulation point to a complex nuclear control mechanism. The presence of catalytic subunit in the nucleus, leading to a CAMP-mediated phosphorylation and conceivable functional modification of nuclear proteins, is most likely of considerable functional consequence resulting in the CAMP-mediated control of nuclear events such as the induction of gene products involved in ovarian differentiation and the ability of granulosa cells to undergo luteinization.
No function of the regulatory subunits at the nuclear level has so far been identified. A report from our laboratory that topoisomerase activity is associated with rat liver regulatory subunit RII (Constantinou et al., 1985) suggests a functional role for RII at the DNA level. However, recent studies in our laboratory have shown that ovarian topoisomerase I activity is not increased after FSH stimulation of estrogen-primed rats.3 Such a FSH-mediated increase would be expected if RII-51 and/or RII-52 possessed topoisomerase activity. Thus, further studies are needed to elucidate the function of ovarian RII variants at the nuclear level. However, because RII-51, -52, and C levels are modulated in nuclei, it is probable that FSH, through cAMP and CAMP-dependent protein kinase, mediates some of its effects in developing preovulatory follicles at the level of the genome through regulation of specific gene expression.