Testicular chromatin activation in hypophysectomized rats

Biochimica et Biophysica Acta, 435 ( 1 9 7 6 ) 1--12 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98601 TESTICULAR CHROMATIN ACTIVATION IN HYPOPHYSECTOMIZED RATS J E N - F U CHIU *, J U D Y T H O M S O N ** a n d L U B O M I R S. H N I L I C A *

Department of Biochemistry, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 (U.S.A.) (Received November 4th, 1975)

Summary Incorporation of labeled thymidine into testicular DNA of hypophysectomized rats began to increase after the administration of testosterone propionate and choriogenic gonadotrophin. While the thymidine incorporation reached maximum in 4 days, the DNA polymerase activity did not culminate until 8 days after the initiation of hormone treatment. The high molecular weight (6--8 S), presumably cytoplasmic DNA polymerase accounted almost entirely for this increase. Administration of testosterone propionate and chorionic gonadotrophin to hypophysectomized rats results in an increase of testicular RNA polymerase and chromatin templating activity. Chain elongation and initiation studies revealed that the increased templating capacity of androgen-stimulated testicular chromatin was almost entirely caused by the increase in the number of initiation sites. While the nuclear polymerase I responded relatively rapidly to hormone stimulation and reached a prominent maximum in about three days, the activity of polymerase II was more sluggish and not as prominent. The in vivo incorporation of ortho[32p]phosphate into chromosomal phosphoproteins occurred early during the androgen treatment and reached a maximum in about 20 h. The protein phosphokinase activity peaked later, approx. 72 h after the first administration of hormones. Introduction Pituitary gonadotrophins are intimately associated with biochemical mechanisms responsible for the production of spermatozoa and stimulation of androgen secretion. After hypophysectomy, a large proportion of type A spermatoP r e s e n t addresses: * D e p a r t m e n t o f B i o c h e m i s t r y , V a n d e r b f l t U n i v e r s i t y S c h o o l o f M e d i c i n e , Nash-

ville0 Tenn. 37232 and ** Department of Biochemistxy, University of Florida, Gainesville, Fla. 32610 (U.S.A.)

gonia and primary spermatocytes degenerate [1,2]. The effect of hypophysectomy can be prevented by repeated administration of luteinizing hormone or testosterone propionate [3]. Among the prominent features of gonadotrophic hormone administration to hypophysectomized animals is the increase of their gonadal size and activation of spermatogenesis in males. Hypophysectomized rats injected with gonadotrophins were reported to increase the DNA polymerase activity of prostate extracts and the [3H] thymidine incorporation rates into DNA of their spermatogonia and spermatocytes [4] and to activate the biosynthesis of enzymes characteristic for spermatid maturation [5]. The aim of the experiments described here was to characterize some of the proliferative and functional responses of hypophysectomized rats treated with chorionic gonadotrophin and testosterone. Materials and Methods Hypophysectomized rats weighing approx. 100 g (Hormone Research Laboratories, Chicago, Illinois) were maintained on commercial laboratory diet (Wyne Laboratory Meal, Allied Mills, Inc.) and 5% aqueous dextrose solution ad libitum. Four weeks after the arrival, the rats were inspected and all animals in which weight and testicular size indicated incomplete hypophysectomy were eliminated. The remaining animals were divided into control and experimental groups and injected daily with 0.1 ml isotonic saline and 0.1 ml olive off (controls) or 5 I.U. of chorionic gonadotrophin (Anuitrin S) in 0.1 ml isotonic saline and 250 mg of testosterone propionate (Savage Laboratories, Inc., Houston, Texas) in 0.1 ml olive oil (experimental). All injections were continued until the day before sacrifice. After decapitation, the testes were rapidly removed, weighed and placed into ice-cold isotonic sucrose solution. Each experimental point consisted of 5--8 rats and represents the average of at least three experiments. Preparation of tissue extracts. All procedures were performed at 4°C except where stated otherwise. The washed testes were homogenized in 5 vol. of 0.25 M sucrose, 5 mM MgC12 and 2 mM 2-mercaptoethanol in glass homogenizer with a loosely fitting teflon pestle (20 strokes), filtered through 8 layers of cheese cloth and centrifuged at 700 × g for 10 min. The supernatant was decanted and processed to represent the cytoplasmic fraction. The nuclear pellet was resuspended in 10 vol. of 2.2 M sucrose, 5 mM MgC12 and 2 mM 2-mercaptoethanol, homogenized and centrifuged at 100 000 X g (max) for 1 h. Nuclei purified in this manner were used for enzymatic assays and for the preparation of chromatin [6]. Incorporation of [Me-3H] thymidine in vivo. Animals were injected intraperitoneally with 50 pCi [Me-3H] thymidine in 0.5 ml saline solution per 100 gm body weight 60 min before sacrifice. Testes were rapidly removed, nuclei isolated and homogenized in 5% cold CC13COOH in ice bath with a glass homogenizer fitted with a teflon pestle. The CC13COOH-insoluble precipitate was spun down and washed three times with 5% CC13COOH at 0°C, once with 95% ethanol, and finally with ethanol/ether (3 : 1). To remove the contaminating RNA, the residue was digested in 0.2 M KOH for 18 hours at 37°C [7]. After this time, 0.05 ml of 70% HC104 were added per each 2 ml of ice-cold 0.2 M

K O H hydrolysate. The precipitate containing D N A was spun down and washed twice with ice-cold C C I 3 C O O H and then dissolved in 0.2 ml N C S (AmershamSearle) for radioactivity measurements. DATA polymerase extraction and determination. Tissue extracts were prepared and centrifuged at 100 000 × g for 1 h. The supernatant from this centrifugation was used as cytoplasmic D N A polymerase source. Nuclear D N A polymerase was extracted from purified nuclei with 0.2 M phosphate buffer p H 7.4 containing 2 m M 2-mercaptoethanol. After centrifugation, at 100 000 × g for 1 h, the supernatant was diaiysed against 0.01 M Tris/HCl buffer, p H 7.4, containing 2 m M 2-mercaptoethanol and used as nuclear D N A polymerase source. The activitiesof D N A polymerases were assayed according to Chiu and Sung [8] with few modifications. The standard reaction mixture contained in 0.4 ml of finalvolume: 2 ~mol Tris/HCl buffer (pH 7.4), 2 #tool MgCI2, 2/~mol dithiothreitol, 20 nmol each of dATP, d G T P and dCTP with 1 #Ci [3H]TTP (50/~Ci/mol) and 20/~g calf thymus native D N A . After incubation at 37°C for 30 rain, the reaction was terminated and the radioactivity was determined [8]. The sucrose density gradient sedimentation patterns were obtained by assaying the D N A polymerase activitiesin extracts of rat testiscytoplasmic and nuclear D N A polymerases. The centrifugation was performed according to the procedure of Martin and Ames [9]. The sample was carefully layered over a sucrose gradient from 5 to 20% (w/v) in cellulose nitrate tubes for Spinco S W 39 L rotor. The samples were centrifuged for 15 h in a Spinco S W 39 L rotor at 36 000 rev./min (0°C). After centrifugation, the bottom of each tube was punctured and 0.2 ml fractions collected. Each fraction was assayed for D N A polymerase activity. Chromatin template activity. The reaction mixture contained in a final volu m e of 250 /~I: 0.2 /~mol each of ATP, G T P and CTP, 0.05 #tool [3H]UTP (spec. act. 125 Ci/mmol), 10/~mol Tris/HCl, p H 8.0, 30/~mol KCI, 0.025 ~mol dithiothreitol, 0.025 #tool E D T A , 0.625 ~mol MnCI2, 5 units of R N A polymerase (prepared from Escherichia coli according to Burgess [10]) and 10 ~g D N A as rat testicular chromatin. The mixture was incubated at 37°C for 10 rain, and the reaction was stopped with 1 ml of ice-cold 10% (w/v) C C I 3 C O O H containing 1% (w/v) sodium pyrophosphate. After five washes with 5 % (w/v) C C I 3 C O O H and 1 % (w/v) sodium pyrophosphate, the precipitated R N A was collected on filter paper (Whatman 3 M M ) discs, washed with C C I 3 C O O H and sodium pyrophosphate solutions, dried with absolute ethanol and counted in toluene-based scintillationcounting fluid. Determination of initiationsites for E. coli R N A polymerase on chromatin templates. The experiments were performed by incorporation of [3H]UTP without re-initiation. The procedure of H y m a n and Davidson [11] was followed as described by Cedar and Felsenfeld [12] with minor modifications. Chromatin samples were incubated with R N A polymerase at 37°C in 0.5 ml of a mixture containing 10 m M Tris/HCl (pH 8.0), 1 mMnCl2, 80 ~ M each of A T P and GTP, and 22/~M [3H]UTP (1000 cpm/pmol). The initiationwas performed at 37°C for 15 rain and was stopped by the addition of 160/~l of 1.6 M (NH4)2SO4. R N A chain elongation was then accomplished by the addition of CTP (80/~M) and MgCl2 (final concentration 5 raM). After 20 rain of incubation in high salt at 37°C, R N A synthesis was stopped by adding 2 ml of 10%

CC13COOH and 1% sodium pyrophosphate. After 15 min at 0°C, the precipitates were collected on Whatman 3 MM filter paper discs. The discs were washed, dried and counted as described above. Since rat DNA contains 54.8% A + T [13], the amount of total nucleoside triphosphates incorporated was assumed to be 3.65 times the incorporation of UTP. For sucrose gradient analysis, 0.2 ml of the final assay mixture were brought to 0.5% in respect to sodium dodecyl sulfate, 7.5 mM EDTA (pH 7.0) and kept at room temperature for 30 min before centrifugation. Analysis of RNA chain length. The chain length of RNA transcribed from chromatin fractions was determined by sucrose gradient centrifugation. 0.2 ml samples were layered over 5 ml of a gradient from 5 to 20% sucrose in 10 mM Tris/HC1, 0.1 M NaC1, 1 mM EDTA (pH 7.9), and centrifuged at 45 000 rev./ min in a Beckman SW 50 rotor at 20°C for 4 h. Fractions (0.2 ml each) were collected and the amount of acid-precipitatable radioactivity in each fraction was determined by reference to the molecular weight markers (mammalian RNAs). The chain length in nucleotides (N) was calculated by the following equation [11,12]: logl0N = 2.10 logl0S20.w + 0.644. The number average chain length (Navg) of the synthesized RNA was determined by the following equation: i Navg

-

~ni i

where ni is the number of RNA chains and Ni is the length of RNA chain in nucleosides. RNA polymerase activities. RNA polymerase activities of hypophysectomized rat testicular nuclei were assayed in a reaction mixture (0.25 ml) conraining 20/~g calf thymus DNA, 10/~mol Tris/HC1 (pH 8.0), 0.2 ~mol MnC12, 0.1 pmol MgC12, 0.2/~mol 2-mercaptoethanol, 1.0 pmol KC1, 0.75/~mol NaF, 0.06 pmol each of GTP, ATP and CTP, 0.01 pmol of UTP, 0.001 pmol of [3H] UTP (spec. act. 2 Ci/mmol), 5 pmol (low salt condition) or 30/~mol (high salt condition) of ammonium sulfate and 100 pg DNA as testicular chromatin. After incubation for 10 min at 37°C, the samples were chilled in an ice bath and 1 ml of 10% CC13COOH in 1% sodium pyrophosphate was added. The acid-insoluble pellet was collected by centrifugation. The pellet was washed with 5 ml 5% CCI3COOH in 1% sodium pyrophosphate three times and dissolved in NCS solubilizer (Amersham-Searle). The radioactivity was determined in a liquid scintillation spectrometer. Labeling of acidic nuclear phosphoproteins in vivo. Carrier-free, ortho[32P]phosphoric acid, neutralized in 0.5 ml of 0.9% NaC1, was injected intraperitoneally into hypophysectomized rats (2 mCi/100 gm of body weight) 60 min before removal of the testes. Isolation and analysis of the phosphoproteins from the isolated nuclei followed the method of Kleinsmith et al. [14] with minor modifications. Nuclei were precipitated with 15% CC13COOH, centrifuged, resuspended in 15% CC13COOH and heated at 90°C for 15 minutes. After cooling in ice, the precipitate was centrifuged, washed three times with 15% CC13COOH, once with chloroform/methanol (1 : 1, v : v), once with chloroform/methanol (2 : 1, v : v) containing 1 ml of concentrated HC1 per 300 ml

of the mixture, and once with ether. The protein residue was finally dissolved in 1.0 M NaOH, an aliquot taken for protein determination, and the remainder of the solution heated at 100°C for 15 min. After cooling, the solution was acidified, the proteins precipitated with silicotungstic acid and the precipitate was extracted with a mixture of isobutanol/benzene (1 : 1, v : v) and aliquots of the extract were mixed with Bray's scintillation modified for determination of 32p activity [8]. The protein phosphokinase activities were determined according to Chiu et al. [15] using casein as a protein substrate. T.he reaction mixture contained 20 #mol of Tris/HC1, pH 7.5, 4 nmol of [8-32P] ATP (0.23 Ci/mmol), 5 pmol of MgC12, 25 pmol of NaC1 and 0.3 mg protein of cytoplasmic preparation or 25 pg DNA as chromatin in a final volume of 0.25 ml. The mixture was incubated at 37°C for 10 min and the reaction was terminated by addition of 10% CC13COOH in 1% aqueous sodium pyrophosphate. The resulting precipitate was washed with cold 5% CC13COOH in 1% sodium pyrophosphate three times and finally dissolved in NCS (Amersham-Searle) solubilizer. The radioactivity of phosphorylated substrates was determined by liquid scintillation counting. Results

While the administration of testosterone propionate and chorionic gonadotrophin did not much change the total body weight of hypophysectomized animals over the experimental period, the net weight of their testes registered a significant increase as early as 24 h after the first hormone injection (Fig. 1). At the same time, the incorporation of [Me-3H] thymidine into the testicular DNA started to rise, reaching a maximum between the third and fourth day of the experiment (Fig. 1). This wave of thymidine incorporation does not parallel the much slower increase in the activity of total testicular DNA polymerase (Fig. 1). The synthesis of cellular DNA proceeds through a number of steps, 0.6

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Fig. 1. T h e e f f e c t o f c h o r l o n i c g o n a d o t r o p h i n and t e s t o s t e r o n e on t h e g r o w t h o f t e s t e s . Male rats were h y p o p h y s e c t o m i z e d 3 0 d a y s b e f o r e t h e b e g i n n i n g of the e x p e r i m e n t . Daffy t r e a t m e n t w i t h h o r m o n e s was c o m m e n c e d a t d a y 30. A t daffy i n t e r v a l s , g r o u p s of 5--8 a n i m a l s were i n j e c t e d inWaperttoneaffy w i t h 5 0 /~Ci of [Me-3H]t h y m i d i n e p e r 1 0 0 g b o d y w e i g h t , and i n c o r p o r a t i o n of l a b e l i n t o n u c l e a r D N A ( A - - - - 4 ) was d e t e r m i n e d 60 rain after the injection. D N A polymerase activity (e ~) w a s m e a s u r e d in g r o u p s o f rats w h i c h w e r e n o t i n j e c t e d w i t h [Me-3H] t h y m i d i n e . T h e t e s t e s w e r e w e i g h e d (~ ~) a n d D N A P o l y m e r a s e w a s e x t r a c t e d and a s s a y e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . Based o n 3 i n d e p e n d e n t e x p e r i m e n t s , t h e v a r i a t i o n s o f D N A s y n t h e s i s w e r e +5---10%, o f t h e t e s t e s w e i g h t + 0 . 5 - - 4 . 4 % a n d of D N A p o l y m e r a s e assays +1---4%.

6

the final result of which is the incorporation of TTP into the final polynucleotide. Of the various enzymes involved, only the activities of cytoplasmic and nuclear DNA polymerases were studied in this paper. These DNA polymerases were isolated and their activities determined at various time intervals of horm o n e treatment. In accord with data in Fig. 1, the cytoplasmic 6--8 S DNA polymerase activity increased slowly, reaching a b o u t 50% increase at the end of the first week and a b o u t 180% increase after 18 days of the treatment. The nuclear 3--4 S DNA polymerase increased only marginally w i t h o u t any significant maximum. The sucrose density gradient centrifugation profiles of the DNA polymerases in control and hormone-treated (18 days) rats are shown in Fig. 2. The increase in DNA synthesis described here occurs approximately at the same time as the activation of chromatin templating activity. The chromatins from hormone-treated animals exhibited higher templating capacity than those of the controls. During the first 24 h after the initiation of hormone treatment, the temptating capacity of testicular chromatin began to rise, doubling in 6 days (Figs. 3 and 4). This rise in templating activity was accompanied by a similar increase in testicular weight. The changes in templating capacity of chromatin may arise either from an increase in the" total number of initiation sites or from a change in the rate of chain elongation. Information a b o u t initiation is obtained from experiments in which only one R N A chain is allowed to be made at each available initiation site. The chain length of the biosynthesized R N A molecules is then determined by sucrose density gradient centrifugation. KNAs templated from either DNA or chromatin under high salt conditions [11,12] had very similar molecular weight distribution. The average size of these ttNAs was between 8 and 10 S. Since these measurements were performed under conditions where the number of R N A chains is equal to the number of initiation sites, both the number of initiation sites as well as the average chain length can be calculated (Table I). In all experiments, the R N A polymerase was in excess in relation to the amount

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Fig. 2. Sucrose density gradient centrifugation of (a) c y t o p l a s m i c a n d (b) n u c l e a r p o l y m e r a s e s i s o l a t e d f r o m h y p o p h y s e c t o m i z e d control r a t s (X X) a n d r a t s t r e a t e d w i t h hormones for 18 days (¢ ~-). A p p r o x . 1 , 5 - - 2 . 0 m g protein of the D N A p o l y m e r a s e p r e p a r a t i o n w a s u s e d in each experiment. A p p r o x i m a t e S values were calculated according to Martin and A m e s [6] using c y t o c h r o m e C and bovine serum a l b u m i n as s t a n d a r d s .

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Fig. 3. I n c r e a s e in t e s t i c u l a z m a s s a n d c h r o m a t i n t e m p l a t i n g a c t i v i t y r e s u l t i n g f r o m h o r m o n e a d m i n i s t r a tion. For three experiments the variations of testicular mass were +0.5--4.4% and of templating activity -+3--7%. Fig. 4. T e m p l a t e a c t i v i t y o f t e s t i c u l a r c h r o m a t i n . H y p o p h y s e c t o m i z e d c o n t r o l r a t s (~ m o n e - t r e a t e d h y p o p h y s e c t o m t z e d r a t s (A "A) (4 d a y s o f h o r m o n e t r e a t m e n t ) .

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of DNA templates. As can be seen in Table I, there was an increase in the number of initiation sites available to E. coli RNA polymerase in chromatin of hormone-treated animals. The average chain length did not change significantly (Table I). The number of initiation sites can also be determined by titration of a fixed amount of RNA polymerase with increasing amounts of DNA template. As shown in Fig. 5 , one unit of E. coli RNA polymerase was treated with increasing amounts of free DNA or chromatin. Control chromatin reached a plateau at about 22 ~g of DNA for 1.65 pmol of the enzyme (based on an asTABLE I D E T E R M I N A T I O N O F T H E N U M B E R O F G R O W I N G C H A I N S BY S U C R O S E G R A D I E N T FUGATION ANALYSIS

CENTRI-

T h e a s s a y c o n d i t i o n s w e r e d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T o t a l n u c l e o t i d e s i n c o r p o r a t e d w e r e calc u l a t e d b y m u l t i p l y i n g t h e U T P i n c o r p o r a t e d b y 3 . 6 5 . T h e a v e r a g e c h a i n l e n g t h w a s d e t e r m i n e d b y sucrose gradient centrifugation. Each sample contained 1.5/~g DNA or 1.5 #g DNA of chromatin. Template

RNA polymerase (units)

UMP incorporated (pmol)

Total nucleotides incorporated (pmol)

Chain length

Initiations (pmol)

DNA

5 10

255 267

931 975

540 550

1.73 1.77

Liver chromatin

5

31

113

540

0.21

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560

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,ug DNA Fig. 5. T i t r a t i o n o f c h r o m a t i n w i t h R N A p o l y m e r a s e . Increasing a m o u n t s o f free or c h r o m a t i n D N A w e r e a d d e d t o 1 u n i t o f E. coU R N A p o l y m e r a s e . A f t e r 2 0 m i n p r o p a g a t i o n in high salt, t h e a m o u n t o f C C l 3 C O O O H - p r e c i ~ i t a t a b l e [ 3 H ] U T P w a s d e t e r m i n e d . D N A : (E -'), h y p o p h y s e c t o m i z e d c o n t r o l (a *) and h o r m o n e t r e a t e d (D D) rat testis c h r o m a t i n .

sumed E. coli RNA polymerase mol.wt, of 4.7 • 105 and a maximum specific activity of 1200 units/mg, ref. 11). The saturation point of chromatin from hormone-treated animals was 14--16 #g of DNA. This further confirms that the higher template activity of hormone-stimulated chromatin principally reflects the increased number of initiation sites. The increased template activity of testicular chromatin in hypophysectomized rats exposed to gonadotrophic hormones is accompanied by the activity of endogenous nuclear RNA polymerase. The RNA polymerase active in low salt assay conditions (polymerase I) increased rapidly during the hormone treatment and slowly declined over the next 8--10 days (Table II). The increase of RNA polymerase II type activity (high salt conditions) was less prominent and reached maximum in 4 days of hormone treatment. The activity in-

T A B L E II RNA POLYMERASE

A C T I V I T I E S IN H Y P O P H Y S E C T O M I Z E D

RAT TESTIS NUCLEI

E x p e r i m e n t a l d e t a i l s are given in Materials and M e t h o d s . T h e s p e c i f i c activities o f R N A p o l y m e r a s e are e x p r e s s e d as p m o l o f [ 3 H ] U M P i n c o r p o r a t e d i n t o R N A / ~ t g D N A . Each value is t h e average o f t w o o r t h r e e n u c l e a r p r e p a r a t i o n s f r o m 5---8 rats e a c h . T h e variation in three e x p e r i m e n t s w a s ± 0 . 4 - - 7 . 9 % . Condition

C o n t r o l rats

E n z y m e a c t i v i t i e s (pmol//~g D N A ) o f rats t r e a t e d w i t h h o r m o n e s for ( d a y s ) 1

2

3

4

5

7

12

L o w salt

0.34

0.41

0.74

1.14

1.05

0.87

0.69

0.51

High salt

0.17

0.19

0.21

0.24

0.30

0.28

0.24

0.15

TABLE III CYTOPLASMIC AND CHROMATIN-BOUND PROTEIN PHOSPHOKINASE ACTIVITIES IN CONTROL AND HORMONE-TREATED RAT TESTES The details of e x t r a c t i o n and assay of protein p h o s p h o k i n a s e s were described in Materials and Methods. The variation in three e x p e r i m e n t s was -+1.3--8.4%. Group of rats

Cytoplasmic kinase 32P i p m o l / m g p r o t e i n

Controls Hormone-treated for

24 48 72 120 288

(h) (h) (h) (h) (h)

C hroma t i n-bound kinase (%)

32p i p m o l / m g p r o t e i n

(%)

5.1

(100)

2.4

(100)

6.8 7.4 9.6 7.1 5.3

(133) (145) (188) (139) (104)

2.7 3.1 3.7 3.4 2.9

(113) (129) (154) (142) (121)

crease of both types of RNA polymerase accompanied the rise in templating activity of testicular chromatin during the first week of h o r m o n e administration (Fig. 3). Essentially, identical results were obtained with a-amanitin. However, because only a limited n u m b e r of assays was performed under these conditions, the incomplete data a r e not included in Table II. Since the phosphorylated chromosomal nonhistone proteins are believed to participate in the regulation of genetic activity [16], the time course of 32p incorporation into the testicular nonhistone protein fraction was investigated in vivo. In hormone-treated animals, the incorporation of 32p into nonhistone proteins rose gradually, beginning at 6 h after h o r m o n e administration, reached a peak at about 20 h and declined slowly to the control levels in about 7 days (Fig. 6). At its maximum, the nuclear nonhistone protein phosphorylation was increased about 70% over the controls. This rise was accompanied by a similar increase in both cytoplasmic and chromatin bound phosphokinase activities

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Fig. 6. In vivo 32p i n c o r p o r a t i o n i n t o n o n h i s t o n e p r o t e i n s of h y p o p h y s e c t o m i z e d c o n t r o l (× ×) and h o r m o n e - t r e a t e d (o o) rats. Activity of the alkali-labile p h o s p h a t e was correlated t o t h e protein c o n t e n t in the acid-insoluble c h r o m a t i n pellet. Each p o i n t represents the average of a t least three independent assays (5--6 rats each). The variations in three e x p e r i m e n t s of h y p o p h y s e c t o m i z e d c o n t r o l s were ±0.5--2% and of h o r m o n e - t r e a t e d rats ±1---6%.

10 (Table III). However, similar to the labeled thymidine-DNA polymerase relationship, the increased incorporation of 32p into the chromosomal nonhistone proteins preceded the considerable rise in phosphokinase activities.

Discussion The primary response of testicular tissues in h y p o p h y s e c t o m i z e d rats to the administration of testosterone and choriogenic gonadotropin is cellular proliferation. The wave of mitotic activity is preceded by a lag period of 36--48 h during which time there is active synthesis of R N A and protein b u t n o t of DNA [ 1 7 - - 2 1 ] . It appears that these biosynthetic processes produce new proteins necessary for the DNA synthesis and cellular proliferation. As would be anticipated, the activation of DNA synthesis was accompanied by an activity increase of DNA polymerase enzymes. In agreement with other investigators who reported that during increased cellular proliferation such as in regenerating rat liver [ 2 2 , 2 3 ] , h e p a t o m a [ 2 3 , 2 4 ] , or other tissues [25,26] only the high molecular weight 6--8 S enzyme activity (a-polymerase) was considerably increased, the testicular 6--8 S DNA polymerase (presumably cytoplasmic) registered a substantial increase. The low molecular weight 3--4 S nuclear enzyme (~-polymerase) did not change significantly in response to t h e h o r m o n e treatment. The anachronism between the thymidine incorporation and 6--8 S DNA polymerase activity maxima is difficult to explain. A similar, although n o t as prominent, shift in 6--8 S DNA polymerase activity was also observed in regenerating rat liver where the peak of D N A synthesis occurred between 24 and 28 h while the highest levels of 6--8 S DNA polymerase activity were not attained until 48 h after h e p a t e c t o m y [ 2 3 ] . A very prominent burst of 6--8 S DNA polymerase activity, apparently n o t associated with comparable DNA replication, was observed by Chiu et al. [23] in rats maintained on hepatocarcinogenic diet. Here, the 6--8 S DNA polymerase experienced a relatively rapid rise around 40 days on the diet which was followed b y a decline to almost normal level within 2 weeks. The 6--8 S DNA polymerase activity increased again at the time of the appearance of hepatomas. Apparently, there is either not a very tight coupling between the activation of 6--8-S DNA polymerases and DNA synthesis or the 6--8-S enzymes represent a multifunctional group. The increase in cell division is preceded by early stimulation of R N A and protein synthesis [ 1 7 - - 2 1 ] . This stimulation coincides with the hormone-induced increase of transcriptional activity of chromatin as well as the activation of endogenous R N A polymerase enzymes. This increase of R N A polymerase initiation sites in hormone-stimulated testicular chromatin described in this paper is comparable with a similar stimulation observed in other systems, e.g. WI-38 human fibroblasts [ 2 7 ] , estradiol-stimulated chick oviduct [28,29], etc. While a considerable stimulation of R N A polymerase activity involves the nucleolar t y p e I polymerase, the chromatin b o u n d enzyme II also registers a significant increase. It can be anticipated that at least a part of this increase reflects the activation o f previously repressed genetic sites required for the synthesis of proteins involved in the preparation for DNA replication and subsequent cell division [ 2 0 , 3 0 , 3 1 ] . Hancock et al. [32] reported that castration decreased, and testosterone

11 administration increased, the activity of "aggregated" RNA polymerase in prostatic nuclei. These authors also found that the effect of testosterone on RNA polymerase was less pronounced when the activity was measured in a high ionic strength medium. Similarly, according to Tata and Widnell [33] the activity of the Mg2+-dependent (low salt) RNA polymerase was stimulated in hypophysect6mized rats 10--12 h after a single injection of triiodothyronine, reaching a peak of 60--90% stimulation at 45 h. The Mn2÷-dependent (high salt) RNA polymerase was not affected at 24 h but its activity increased by 30--40% at 45 h. Our experiments with testicular chromatin of hypophysectomized rats show a similar lag of the ammonium sulfate (high salt) stimulated enzymes (polymerase II). Apparently the hormone-depleted target cells react to a sudden supply of hormone first by increasing their protein-synthesizing capacity and then by a more selective activation of specific transcription sites in chromatin. This activation is preceded by a wave of nuclear protein phosphorylation. Numerous data in the literature point to the more than casual relationship between the phosphorylation of chromosomal nonhistone proteins and gene stimulation [16]. Indeed, it was suggested that the phosphorylation of nuclear proteins may be a necessary step in the activation of selected genes [16]. Ahmed and associates [33--35] have shown that rat ventral prostate nuclei in vitro can incorporate the label from [~,.32p] ATP into chromosomal nonhistone proteins and that such incorporation can be correlated with the increased transcriptional activity in the prostate following hormonal treatment of orchiectomized rats. In their more recent work, Ahmed and Wilson [35] demonstrated the presence of androgen-sensitive protein phosphokinase associated with rat ventral prostate chromatin which may modulate the phosphorylation of chromosomal nonhistone proteins mediated by testosterone in this target tissue. These results are comparable to our data on the hormonal activation of testicular protein phosphokinases and increase of chromosomal nonhistone protein phosphorylation. Based on our previous and present observations, as well as on the transcriptional-activation data, we would like to emphasize the temporal relationship between nuclear protein phosphorylation and the activation of schedule of chromatin. The administration of testosterone and chorionic gonadotrophin first activates chromosomal protein phosphorylation and the corresponding enzymes. This most likely affects the structure of chromatin, allowing the exposure or derepression of specific genes (increase in RNA initiation sites) with a concomitant increase of RNA polymerase activity. After the biosynthesis of specific proteins which are probably essential for cellular proliferation, the DNA synthesis is activated along with the mobilization of appropriate enzymes. It is difficult, at the present time, to present a definite account of testicular cells involved in the biochemical changes described here. Because of the effects of chorionic gonadotrophins, the Leydig cells may account for a considerable portion of the reported increase in DNA synthesis. The activation of transcriptional activity and nuclear protein phosphorylation can be, on the other hand, primarily associated with the cellular elements inside the seminiferous tubules. Recently improved techniques for testicular cell fractionation should permit a more detailed extension of experiments described in this paper.

12 Acknowledgements This research was supported by U.S. Public Health Service Grant CA 07746 and Robert A. Welch Foundation Grant G 138. References 1 Steinberger, E. (1971) Physiol. Rev. 51, 1--22 2 Clermont, Y. and Morgentaler, H. (1955) Endocrinology 5 7 , 3 6 9 - - 3 8 2 3 Leblond, C.P., Steinberger, E. and Roosen-Runge, E.C. (1963) in Mechanisms Concerned with Conception (Hartmen, E.G., ed.), pp. 1--22, Pergamon Press, Oxford 4 0 r t a v a n t , R., Courot, M. and Hochereau-De Reviers, M.T. (1972) J. Reprod. Fertil. 31,451---453 5 Males, J.L. and Tur kington, R.W. (1971) Endocrinology 68, 579--588 6 Wilhelm, J.A., Ansevin, A.T., Johnson, A.W. and Hnilica, L.S. (1972) Biochim. Biophys. Acta 272, 220--230 7 Schmidt, G. and Tannhauser, S.J. (1945) J. Biol. Chem. 161, 83--89 8 Chin, J.F. and Sung, S.C. (1970) Biochlm. Biophys. Acta 209, 34--42 9 Martin, R.G. and Ames, B.N. (1961) J. Biol. Chem. 236, 1372--1379 10 Burgess, R.R. (1971) J. Biol. Chem. 244, 6 1 6 0 - - 6 1 6 7 11 H y m a n , R.W. and Davidson, N. (1970) J. MoL Biol. 50, 421--438 12 Cedar, H. and Felsenfeld, G. (1973) J. Mol. Biol. 7 7 , 2 3 7 - - 2 5 4 13 Woodhouse, D.L. (1954) Biochem. J. 5 6 , 3 4 9 - - 3 5 2 14 Kleinsmith, L.J., Allfrey, V.G. and Misky, A.E. (1966) Proc. Natl. Acad. Sci. U.S. 55, 1182--1189 15 Chin, J.F., Craddock, C., Getz, S. and Hniliea, L.S. (1973) FEBS Lett. 33, 2 4 7 - - 2 5 0 16 Stein, G.S., Spelsberg, T.C. and Kleinsmith, L.J. (1974) Science 183, 817--824 17 Hechter, O. and Halkerston, I.D. (1964) in The H o r m o n e s (Pincus, G., Thimann, K.V. and Astwood, E.B., eds.), Vol. 5, pp. 697--825, Academic Press, New Y o r k 18 Liao, S. and Fang, S. (1970) Vitam. Horm. 27, 17--90 19 Grant, J.K. (1969) Essays Biochem. 5, 1--58 20 O'Malley, B.W., McGuire, W.L., Kohler, P.O. and Korenma n, S.G. (1969) R e c e n t Progr. Horm. Res. 25, 1 05--160 21 Williams-Ashman, H.G. and Reddi, A.H. (1971) Annu. Rev. Physiol. 33, 31--72 22 Chang, L.M.S. and Bollum, F.J. (1972) J. Biol. Chem. 247, 7 9 4 8 - - 7 9 5 0 23 Chiu, J.F., Craddock, C., Morris, H.P. and Hnilica, L.S. (1974) Cancer Biochem. Biophys. 1, 13--21 24 Ove, P., Laszlo, J., Jenkins, M.D. and Morris, H.P. (1969) Cancer Res. 29, 1557--1561 25 Chiu, J.F. and Sung, S.C. (1972) Biochim. Biophys. Acta 2 6 9 , 3 6 4 - - 3 6 9 26 Weissbach, A. (1975) Cell 5, 101--108 27 Hill, B.T. and Baserga, R. (1974) Biochem. J. 141, 27--34 28 Cox, R.F., Haines, M.E. and Garey, N.H. (1973) Eur. J. Biochem. 32, 513--524 29 Tsal, M.J., Schwartz, R.J., Tsal, S.R. and O'Malley, B.W. (1975) J. Biol. Chem. 250, 5165--5174 30 Stein, G.S. and Baserga, R. (1970) J. Biol. Chem. 245, 6097---6105 31 Stein, G.S., Chaudhuri, S. and Baserga, R. (1972) J. Biol. Chem. 247, 3 9 1 8 - - 3 9 2 2 32 Hancock, R.L., Zelis, R.F., Shaw, M. and Williams-Ashman, H.G. (1962) Biochlm. Biophys. Acta 55, 257--260 33 Tata, J.R. and Widnell, C.C. (1966) Biochem. J. 9 8 , 6 0 4 - - 6 2 0 34 Ah med , K. (1971) Biochim. Biophys. Acta 243, 38--48 35 A h m e d , K. and Ishida, H. (1971) Mol. Pharmacol. 7 , 3 2 3 - - 3 2 7 36 Ah med , K. and Wilson, M.J. (1975) J. Biol. Chem. 250, 2 3 7 0 - - 2 3 7 5