Brain Research, 94 (1975) 99-113

99

~) Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

T H E M E T A B O L I C C L E A R A N C E RATE, H E A D A N D BRAIN EXTRACTIONS, A N D BRAIN D I S T R | B U T I O N A N D METABOLISM OF P R O G E S T E R O N E IN THE ANESTHETIZED, FEMALE MONKEY (MACACA MULATTA)

R. B. BILLIAR, B. LITTLE, I. KLINE, P. REIER*, Y. TAKAOKA ANDR. J. WHITE Department of Reproductive Biology, Case Western Reserve University, and (Y.T. and R.J.W.) Brain Research Laboratories, Division of Neurosurgery, Cleveland Metropolitan General Hospital and Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.)

(Accepted March 1lth, 1975)

SUMMARY The brain distribution and metabolism of progesterone were studied in female, rhesus monkeys. Adult monkeys were anesthetized with ketamine and were given a constant infusion of [3H]- or [14C]progesterone. Blood samples were obtained from cannulae inserted into the carotid artery, the jugular vein and lateral (transverse) sinus. The metabolic clearance rate of progesterone was 295 ± 49 (S.E.) l/day. The head extraction of progesterone was 30.4 ~: 8.3 ~ (S.E.) and the brain extraction 26.0 ± 9.1 ~ (S.E.). The peripheral conversion ratios of progesterone to 20a-hydroxypregn-4en-3-one (20a-OHP) and 5a-pregnan-3,20-dione (5a-DHP) were 10.0 ± 1.3 ~,, (S.E.) and 2.4 ± 0.3 ~ (S.E.), respectively. These same conversion ratios for across the head were 4.8 zk 1.0 °/o (S.E.) and 1.5 :k 0.6 ~ (S.E.) and for across the brain 5.0 -[_ 0.7 (S.E.) and 2.2 _k 0.6 ~ (S.E.). The concentration of radioactive progesterone was 2-5 times higher in brain tissues compared to the carotid arterial blood. The tissue concentrations of radioactive progesterone compared to a cerebrum 'control' sample: was lower in the central gray (P < 0.05); were the same for the amygdala, hippocampus, preoptic-anterior hypothalamus, cerebellum, hypothalamus, thalamus, and anterior pituitary; and were higher in the cerivcal spinal cord, optic chiasm, mesencephalon, medulla oblongata and pons (P < 0.1). The distribution pattern of 20a-OHP formed from progesterone was similar to that of progesterone. 5a-DHP formed from progesterone had a different distribution than progesterone, being highest in the central gray area. High concentrations of 5a-DHP were also observed in the mesencephalon, medulla oblongata and hypothalamus and low values in the anterior pituitary. Infusions of [3H]20a-OHP and [3H]5a-DHP were used to evaluate the in vivo metabo* Present address: Laboratory of Neuropathology and Neuroanatomical Science,N.I.N.S.S., N.I.H., Bethesda, Md. 20014, U.S.A.

I00 lism of progesterone by different brain areas. [3H]Estradiol infused into one monke~ had its highest concentration in the anterior pituitary which was 20 times higher than in the carotid arterial blood.

INTRODUCTION

Progesterone can affect reproduction in the primate. Under certain conditions it increases circulating gonadotropin in womenZT,ss, but progesterone can also inhibit the estradiol induced luteinizing hormone (LH) surge in the monkey1°!4°. Progesterone also inhibits female sexual behavior in the monkey24. Although such observations suggest, but do not prove, a direct action of progesterone in the brain, the site(S) and mechanism(s) of action of progesterone in the monkey brain are not presently known. In the human there is an appreciable extrasplanchnic clearance ofprogesteronelL It is not known what contribution the brain makes to the extrasplanchnic clearance of progesterone in the primate but a significant proportion (10 ~) of the cardiac output goes to the brain. Studies in sheep indicate that there is an appreciable extraction (25~) of progesterone and an active conversion of progesterone to 20a-hydroxypregn-4-en-3-one (20a-OHP) by the head 3. In the present report the head and brain extractions and the brain distribution and metabolism of progesterone were studied in anesthetized rhesus monkeys by the continuous infusion methodL Cephalic and cerebral arterial and venous concerttrations of radioactive progesterone and two of its metabolites, 20a-OHP and 5apregnan-3,20-dioae (Sa-DHP), were determined and also their concentrations in various areas of the brain. The continuous infusion method has the advantage over the single injection method as considerably fewer animals need to be used. In single injection studies numerous time samples are required for construction of meaningful uptake and distribution patterns 5. METHODS

Adult, female rhesus (Macaca mulatta) monkeys were anesthetized with ketamine CI-716 and CI-634 (Parke-Davis), tracheally intubated, and maintained in a heparinized state. Blood pressure and heart rate were continuously monitored with an indwelling femoral catheter connected to a pressure transducer and a direct write-out Grass recorder. Radioactive steroids were infused through a Teflon cannula into a femoral vein. Blood collections were made from polyethylene cannulae positioned at the carotid bifurcation (inserted via the external carotid artery), from the opposite jugular vein, and from the lateral (transverse) sinus of the brain. [1,2-all]Progesterone, [4-14C]progesterone, [t,2-aH]20a-hydroxypregn-4-en-3one, [1,2-aH]5a-pregnan-3,20-dione, and [2,4,6,7-aH]estradiol were purchased from New England Nuclear Corporation and purified by thin-layer chromatography (LTC). Radioactive steroids were infused in 30 ml of 10% ethanol-saline (v/v) at a rate of about 0.1 ml/min into the femoral vein. At 110, 160 and 210 min after the start of the

101 infusion blood samples of 1.5 ml were drawn from the carotid artery, jugular vein, and lateral sinus after first withdrawing 2-3 ml. The first 2-3 ml were returned to the animals via the jugular vein cannula. The cranium was carefully removed (about 1520 min) and blood samples again drawn. Then, while cerebral circulation was still intact, the brain was rapidly removed surgically. One milliliter aliquots of blood were added to the following steroids for estimation of procedural losses: 10/~g of [14C]progesterone, 100 #g [14C]20a-OHP and 200/~g 5a-DHP for experiments with infusions of [3H]progesterone; 100 ~g progesterone, 100 #g 20a-OHP and 2(30/~g 5a-DHP for the experiments with [14C]progesterone and [ZH]20a-OHP or [3H]5a-DHP; and 1(30 #g [3H]progesterone, 100/~g [3H]20a-OHP, 2130 /~g [3H]5a-DHP, 200/~g [14C]estradiol and 2(30 #g [14C]estrone for the experiment with [14C]progesterone and [aH]estradiol. These steroids were suspended in 0.1 ml ethanol and 1.0 ml water. The blood samples were immediately frozen. Neural tissue samples were obtained in the following order: anterior and posterior pituitary, optic chiasm, preoptic-anterior hypothalamic (MPO-AH) region, temporal cerebral cortex, hypothalamus, thalamus, central gray, mesencephalic-tegmentum, pons, medulla, cerebellar cortex (vermix), and cervical spinal cord. For isolation of the MPO-AH region, a section was made which extended from a mid-chiasmatic level in line with the anterior commissure to the caudal margin of the optic chiasm. The region between the chiasm and anterior commissure was removed and trimmed laterally at a point approximating the interface of the medial and lateral preoptic areas. The hypothalamus was then isolated by cutting at the caudal edge of the mammillary bodies, dorsally in line with the anterior commissure and laterally on the medial border of the cerebral peduncles; the rostral limit was defined by the first section made in obtaining the MPO-AH sample. Specimens of central gray, mesencephalictegmentum, ports, medulla and cervical spinal cord were obtained from free-hand transverse sections approximately 2-3 mm thick. Central gray and mesencephalictegmentum samples were obtained from a section made at the level of the superior colliculus. The brain samples were weighed in random order and placed in 10 ml acetone which contained the same recovery steroids as for blood. About 20 rain elapsed from time of removal of the brain to placement of the last sample in acetone. A sample of abdominal fat was also placed in acetone. A sample of the uterus was placed in formalin for histological analysis. Tissue samples were stored at --20 °C. After thawing, blood samples were immediately extracted twice with 5 ml benzene and once with 2 ml benzene. The pooled benzene extracts were dried under a nitrogen stream at 45 °C. Tissue samples were homogenized in acetone and then centrifuged at 113(30rev./min for 15 min. The supernatants were taken to dryness and the residues were dissolved in 5 ml benzene and stored at 4 °C overnight. The extracts containing a white precipitate were filtered through glass-wool, and the filtrate was taken to dryness and dissolved in 5 ml heptane. Five milliliter of 9 0 ~ methanol10 ~ water (v/v) was added, and the solutions vortexed for 1 rain followed by centrifugation. The methanol layer was used for analysis. Radioactive steroids were isolated from the blood and tissue extracts by TLC.

102 For infusion studies of [3H]progesterone the extracts were first chromatographed in benzene-acetone (100:15, v/v). The areas corresponding to 5a-DHP, progesterone and 20a-OHP were eluted with methylene dichloride-methanol (9:1, v/v; used for all elutions from TLC). 5a-DHP was then chromatographed in hexane-ethyl acetate (3 : 1, v/v), and the 5a-DHP eluted and taken to dryness. Two milliliters of methanol were added, the samples were placed at 4 °C overnight, warmed to room temperature and 0.2 ml portions in duplicate were placed in tubes. The remaining 1.6 ml was transferred to scintillation vials, taken to dryness and the radioactive 5a-DHP was quantified by liquid scintillation spectrometry, To the 0.2 ml samples were added 0.2 ml 8 N methanolic KOH and 0.2 ml of 0.5 % dinitrobenzene in methanol (both from Sigma Chemical Company). The reaction mixture was transferred to microcuvette (! cm light path) and covered with aluminum foil. After 10 rain the optical density was recorded at 520 nm and the procedural losses of 5a-DHP were calculated. After the first TLC progesterone was chromatographed in benzene-ethyl acetate (100:22.5, v/v), eluted, taken to dryness and the residue acetylated with acetic anhydride-pyridine (1:4). The progesterone was then chromatographed in benzeneacetone (100:15, v/v), the progresterone eluted into scintillation vials, taken to dryness and radioactive progesterone quantified by liquid scintillation spectrometry. Procedural losses were calculated from the remaining disint./min of the added [14C]progesterone. After the first TLC, 20a-OHP was chromatographed in chloroform-methanol (100:1, v/v), eluted, taken to dryness and acetylated. The 20a-OHP acetate was then chromatographed in benzene-acetone (!t30:15, v/v), eluted into scintillation vials, taken to dryness and the 14C and aH quantified by liquid scintillation spectrometry. Procedural losses were calculated from the disint./min of 14C. The radicchemical purity of the progestogens isolated by these procedures was confirmediby'crystallization (results not shown). In the experiment involving infusion of [14C]progesterone and [ZH]20a-OHP, 5a-DHP, progesterone and 2Qa-OHP were isolated as described above and procedural losses for 5a-DHP were estimated as described. For quantification of progesterone and 20a-OHP after the last TLC, nine-tenths of the sample was used for quantification of radioactivity by liquid scintillation spectrometry and one-tenth of the sample was dissolved in methanol and used for estimation of radio-inert steroid by recording the optical density at 230, 240 and 250"nm and comparing with appropriate standards to correct for procedural losses. In the infusion experiment with [14C]progesterone and [aH]5a-DHP the isolation and procedure for estimating procedural losses were the same as with the [14C]progesterone and [ZH]20a-OHP experiment except that an additional TLC chromatographic step of hexane-ethyl acetate (3:1, v/v) was used for progesterone. For the [14C]progesterone and [3H]estradiol study the extracts were first cbromatographed in chloroform-methanol (99:1, v/v). Three areas were eluted. One contained 5a-DHP and progesterone, the second estrone and 20a-OHP, and the third estradiol. Progesterone and 5a-DHP were isolated by the chromatographic sequence used for their isolation in the [all]progesterone experiments described above except

103 TABLE I METABOLICCLEARANCERATEAND HEADAND BRAIN EXTRACTIONSOF PROGESTERONEIN FEMALEMONKEYS

Monkey Stage of cycle*

2 3 6

Early luteal Midcycle Early follicular

7

Midluteal

8

Late follicular

S.E. : t

Steroid infused

[3H]Progesterone pH]Progesterone [14C]Progesterone pH]20a-OHP [14C]Progesterone [ZH]Estradiol [14C]Progesterone [aH]5a-DHP

Progesterone Metabolic Head clearance rate extraction** (1~day) (%)

Brain extraction*** (%)

402 327 281

23.1 25.4 26.5

19.8 17.7 17.7

116

62.5

62.2

349

14.4

12.7

295 49

30.4 8.3

26.0 9.1

* Stage of cycle was determined by uterine histology. ** Head extraction was estimated from the difference between the concentrations of radioactive progesterone in the jugular and carotid bloods. *** Brain extraction was estimated from the differences between the concentrations of radioactive progesterone in the lateral sinus and the carotid bloods.

that procedural losses were corrected by comparison with the disint./min of added [all]progesterone and [3H]5a-DHP. 20a-OHP was separated from estrone by chromatography in chloroform-ethyl acetate (4:1, v/v). The 20a-OHP was then chromatographed in chloroform-methanol (ll30:1, v/v), eluted, taken to dryness, acetylated and 20a-OHP acetate chromatographed in benzene-acetone (1130:15, v/v). [3H, 14C]20a-OHP acetate was quantified by liquid scintillation spectrometry. Procedural losses were corrected by disint./min of 3H. After chromatography in CHCla-ethyl acetate, the estrone was eluted, taken to dryness, acetylated and chromatographed in heptane-ethyl acetate (7:3, v/v). [3H, 14C]estrone acetate was quantified by liquid scintillation spectrometry and procedural losses were determined by the [14C] disint./min. After the first chromatography, estradiol was chromatographed in CHCl3-ethyl acetate (4:1, v/v), eluted, taken to dryness, acetylated and chromatographed in heptane-ethyl acetate (7:3, v/v). [all, 14C]Estradiol diacetate was quantified by liquid scintillation spectrometry and procedural losses calculated by comparison with the disint./min of added [14C]estradiol. The metabolic clearance rates, peripheral conversion ratios and transfer constants of the steroids were calculated by the method of Baird et al. 2. Transtissue transfer constants, transtissue conversions and intratissue conversions were calculated by the formulations of Chapdelaine v. RESULTS

The blood metabolic clearance rate (MCR) of progesterone in 5 female monkeys

104 TABLE II MONKEY BRAIN DISTRIBUTION OF PROGESTERONE A N D

20a-OHP

AND

5a-DHP

FORMED FROM PRO-

GESTERONE

Tissue

Central gray Amygdala Hippocampus Preoptic-anterior hypothalamus Cerebrum Cerebellum Hypothalamus Remaining pituitary** Spinal cord Thalamus Anterior pituitary Optic chiasm Mesencephalic-tegmentum Medulla oblongata Pons Fat

n

Progesterone. Ratio to concentration o f

20a-OHP

Cerebrum

Carotid blood

Ratio to cerebrum

5a-DHP ...... Ratio to cerebrum

4 2 2

0.66 4- 0,03* 0.77 0.89

1.56 4- 0.24 1.74 1.75

0.39 4- 0.09* 1.02 0.99

7.23 :~: 3.66* 0.27 0.97

5 5 5 5 5 5 4 5 5 5 5 5 4

0.94 4- 0.05 1.00 1.07 ± 0.10 1.11 4- 0.11 1.29 ± 0.51 1.69 4- 0.28* 1.69 4- 0.20 1.73 ~ 0.59 1.82 4- 0.24* 1.83 4- 0.17' 1.86 4- 0.39* 1.87 ~ 0.25* 1.61 ± 1.37

2.07 2.18 2.22 2.17 2.34 3.36 4.07 3.12 3.78 4.08 3.56 3.66 4.64

0.43 4- 0.06* 1.00 0.51 4- 0.05* 0.55 4- 0.10" 0.43 4- 0.05* 1.42 4- 0.18 0.95 4- 0.15 0.35 4- 0,05* 1.26 4- 0.22 0.83 4- 0.10* 1.47 4- 0.21 1.40 4- 0.15 0.58 4- 0.45

2.23 :L 1.14 1.00 2.34 2~ 0.34 * 4.32 .L 2.93 1.12 :~: 0~86 3.32 ± 1.81 1.75 :=: 0.39* 1.29 .L 0.91 1.72 :J: 0.88 4.78 ± 2.63* 4.66 ~-: 2,98 3.82 j: 1.66~ 0.59 :~ 0.58

4- 0.45 4- 0.44 4- 0.36 4- 0.46 4- 0.53 4- 0.47 ± 0.79 ± 0.56 4- 0.68 4- 0.92 t 0.44 :~ 0.38 ~ 4.15

* Significantly different from the cerebrum at the 0.10 level or less as calculated by the paired t-test from disint./min/mg concentrations. ** In this and the following Tables 'remaining pituitary' refers to the pituitary sample left after removal of the anterior pituitary. It consists primarily of the posterior pituitary but some anterior pituitary may be included.

was 295 4- 49 (S.E.) l / d a y (Table I). One m o n k e y (No. 7) h a d a low, b u t c o n s t a n t (coefficient o f v a r i a t i o n 12 ~o) M C R c o m p a r e d to the o t h e r 4. T h e r e was a positive e x t r a c t i o n ( a r t e r i o v e n o u s difference) o f p r o g e s t e r o n e b o t h b y the h e a d o f 30.4 =r8.3 ~ (S.E.) a n d the b r a i n 26.0 4- 9.1 ~o (S.E,) a n d this positive e x t r a c t i o n was observed in each a n i m a l a l t h o u g h No. 7 h a d u n u s u a l l y high h e a d b r a i n extractions o f 62.5 a n d 62.7 ~ , respectively. The h i g h e x t r a c t i o n s in m o n k e y No. 7 were c o n s t a n t t h r o u g h o u t the s a m p l i n g time (coefficient o f v a r i a t i o n s o f 7 a n d 1 1 ~ , respectively). P a r t o f the e x t r a c t i o n o f p r o g e s t e r o n e by the h e a d a n d b r a i n o f the m o n k e y c a n be a c c o u n t e d by the m e t a b o l i s m o f p r o g e s t e r o n e to 2 0 a - O H P a n d 5 a - D H P . The peripheral c o n v e r s i o n r a t i o 2 o f p r o g e s t e r o n e to 2 0 a - O H P was 10.0 2 : 1 . 3 ~ (S.E.) and to 5 a - D H P 2.4 4- 0.3 ~ (S.E.). T h e c o n v e r s i o n o f p r o g e s t e r o n e to 2 0 a - O H P across the h e a d (based o n c a r o t i d a n d j u g u l a r b l o o d values) was 4.8 ~ 1 . 0 ~ (S.E.) a n d for the b r a i n (based on c a r o t i d a n d l a t e r a l sinus b l o o d values) 5.0 :z 0.7 ~ (S.E.) which were n o t significantly different. The c o r r e s p o n d i n g values for the c o n v e r s i o n o f p r o g e s t e r o n e to 5 a - D H P were 1.5 4- 0.6 ~ (S.E.) for the head a n d 2.2 :E 0.6 ~ (S.E.) for the brain ( P - - 0.10). The conversions across the h e a d a n d b r a i n are net (minimal) values since they h a v e n o t been c o r r e c t e d for the h e a d a n d b r a i n extractions o f 2 0 a - O H P and 5a-

105 TABLE III MONKEY BRAIN DISTRIBUTION OF [3H]ESTRADIOL DURING CONTINUOUS INFUSION OF [3HIEsTRADIOL AND [14C]PROGESTERONE

Tissue

L3H] Estradiol Ratio to cerebrum concentration

Amygdala 0.97 Cerebrum 1.00 Cerebellum 1.22 Hippocampus 1.64 Preoptic-anterior hypothalamus 1.68 Thalamus 1.79 Central gray 1.99 Hypothalamus 2.26 Optic chiasm 2.52 Mesencephalic-tegmentum 2.81 Pons 3.16 Spinal cord 4.11 Medulla oblongata 4.81 Remaining pituitary** 6.49 Anterior pituitary 39.7 Fat 1.08

Ratio to carotM blood concentration

0.54 0.56 0.67 0.90 0.93 0.98 1.09 1.24 1.39 1.55 1.74 2.26 2.65 3.58 21.9 0.59

Ratio o f [3H]estrone to [3H]estradio l concentration (%)

4.2 0 9.0 3.7 4.6 12.6 4.8 9.5 6.1 12.4 12.3 9.2 11.8 15.3 3.3 --

Ratio o f [aH]estradiol to z.14C]pro_ gesterone*

0.61 0.46 0.39 0.69 0.86 0.54 1.29 0.86 0.87 0.70 0.70 0.68 0.66 0.92 4.57 1.41

* Calculated from the ratio of [aH]estradiol and [t~C]progesterone tissue concentrations to the [3H]estradiol and [14C]progesteronecarotid arterial blood concentrations. ** See legend of Table II.

DHP. Similarly the progesterone extractions listed in Table I are net values since they have not been corrected for the head and brain conversions of radioactive metabolites, e.g., 20a-OHP, to progesterone and release of this 'newly' formed radioactive progesterone into the jugular vein and lateral sinus. Most brain areas and the anterior pituitary had concentrations of radioactive progesterone greater than the concentrations in the carotid arterial blood but the concentrations in all areas of the brain were less than twice that of the cerebrum (Table 11). The concentration of radioactive progesterone in the central gray was significantly (P < 0.05) lower than that present in the cerebrum. The spinal cord (cervical), optic chiasm, mesencephalon, pons and medulla oblongata had higher (P < 0.1) concentrations than the cerebrum while the other areas sampled did not. To test the effect of anesthesia and other unknown variables on the brain distribution of steroids, one monkey was given a continuous infusion of [aH]estradiol as well as [14C]progesterone. The concentration of [aH]estradiol in the anterior pituitary is 40 times that of the cerebrum and 20 times that of the carotid blood (Table III). The hypothalamic concentration of [aH]estradiol is twice that of the cerebrum but equal to that of the carotid arterial blood. Many areas other than the hypothalamus also have a higher concentration of [aH]estradiol compared to the

106 carotid blood. This distribution pattern of estradiol in the monkey brain is similar to that observed in the unanesthetized rat by the single injection technique TM. The M C R of estradiol was 187 1/day (C.V. 4.3 ~/o ~) and the head and brain extractions of estradiol were 32.1 ~J'0(C:V. 15 ~o) and 39.3 ~ (C.V. 9.9 ~), respectively. The head and brain extractions (Table I) of progesterone and the conversion of progesterone to 20a-OHP and 5a-DHP suggested that the monkey brain could actively metabolize progesterone. Radioactive 20a-OHP, formed from progesterone, could be isolated from every brain area sampled (Table II). The distribution of the 20a-OHP formed from progesterone was similar to the brain distribution of progesterone although the cerebral concentration was higher for 20a-OHP than for progesterone. To determine whether the 20a-OHP isolated from a brain sample was formed outside of the brain and concentrated in the brain or whether the 20a-OHP was formed directly in the brain tissue itself, the tissue distribution of 20a-OHP and its formation from progesterone were studied in one monkey which was given a continuous infusion of [3H]20a-OHP and [14C]progesterone. The MCR of 20a-OHP was 262 I/day (C.V. 29 ~ ) and the peripheral transfer constant 2 of progesterone to 20a-OHP, ~P20a ~)BB , was 0.073 (C.V. 8.2 ~/o) and the peripheral transfer constant of 20a-OHP to progesterone, (_'~20tIP ~'~aa , was 0.012 (C.V. 17~o). The head and brain extractions of progesterone, corrected for metabolism of 20a-OHP back to progesterone, (1 -~AV] ,~eP~7 were 26.5 ~o and 17.7 ~ , respectively, and the head and brain extractions of 20a-OHP, corrected for metabolism of progesterone back to 20a-OHP, (1-t~2~TM) were 12.4 ~'~i and 1.9~o, respectively. The transhead and transbrain conversions of progesterone to 20a-OHP, corrected for extraction of 20a-OHP, (e~v°a)7 were 3.5 ~,i and 5.5 ~,;, respectively. The transhead and transbrain conversions of 20a-OHP to progesterone, (_20aP~] were 0.9 ~ and 0.4 ~ , respectively. corrected for extraction of progesterone, ~'Av The brain distributions of [3H]20a-OHP and [14C]progesterone were similar (Table IV). To calculate the intratissue interconversions of progesterone and 20a-OHP it has to be assumed that the radioactive concentrations of the 2 steroids delivered to each tissue are the same as in the carotid arterial blood, that the tissue radioactive concentrations of the steroids represent only tissue concentrations devoid of steroids in the intercellular water, and that the entire pool of radioactive steroid is accessible to tissue metabolism 7. Brain metabolism thus favors the conversion of progesterone to 20a-OHP over the conversion of 20a-OHP to progesterone (Table IV), as opposed to that observed for adipose tissue. Most of the 20a-OHP is formed in the brain tissues rather than being formed in the periphery and stored in the brain. The highest concentration of radioactive 5a-DHP formed from progesterone was in the central gray whereas this tissue had the lowest concentration of progesterone and 20a-OHP (Table If). The hind brain, hypothalamus and mesencephalon also had high concentrations of this metabolite. The high tissue :carotid blood ratios of this metabolic suggested that the 5a-DHP was being formed in the brain tissues from progesterone. This was confirmed by infusing a monkey with [3H]5a-DHP and [t4C]progesterone (Table V). The M C R of 5a-DHP was 600 I/day (C.V. 9,7 %) and the peripheral conversion of progesterone to 5a-DHP, (t))BB, ~ ,vsa was 0.054 (C.V. 17 %). The corrected head and brain ' extractions • 5asa (I-QA v ) were 35.9 ~ and 34.8 ~ , respective-

107 TABLE IV MONKEY BRAIN DISTRIBUTION OF

[aH]20a-OHP, AND 20a-OHP AND PROGESTERONE TISSUE [3H]20a-OHP AND [14C]PROGESTERONE

INTERCON-

VERSIONS D U R I N G A CONTINUOUS INFUSION OF

OPe0,~ and T'2

20at are ~TT

the intratissue transfer constants of progesterone to 20a-OHP and 20a-OHP to

progesterone, respectively, and were calculated according to Chapdelaine 7.

Tissue

Central gray Preoptic-anterior hypothalamus Anterior pituitary Cerebellum Cerebrum Hypothalamus Thalamus Remaining pituitary** Optic chiasm Spinal cord Mesencephalic-tegmentum Medulla oblongata Pons Fat

Ratio of

[3H]2Oa-OHP Ratio to cerebrum concentration

Ratio to carotM blood concentration

0.70 0.81 0.89 0.90 1.00 1.11 1.33 1.41 1.50 1.52 1.52 1.53 1.84 --

1.66 1.92 2.10 2.12 2.36 2.61 3.14 3.32 3.55 3.59 3.60 3.62 4.35 0.20

~TT2OcIP ~TT20Clt"

i :~H]2Oa-OHP to 114C)progesterone*

1.02 0.74 0.68 0.82 1.01 0.79 0.60 1.29 0.71 1.06 0.64 0.89 1.02 1.25

0.095 0.068 0.033 0.129 0.290 0.212 0.153 0.093 0.200 0.226 0.090 0.218 0.159 0.001

0.124 0.056 0.031 0.009 0.068 0.031 0.023 0.137 0.045 0.020 0.032 0.015 0.007 0.547

* Calculated from the ratio of [aH]20a-OHP and [~zC]progesterone tissue concentrations to the [aH]20a-OHP and [14C]progesterone carotid arterial blood concentrations. ** See legend of Table II.

ly, and the corrected transhead and transbrain conversions of progesterone to 5a-DHP (~Av-eSa'~were 3.7 ~ and 3.4 ~ , respectively. The brain distribution pattern of [3H]5aD H P was similar to that of [14C]progesterone except that the anterior pituitary had relatively more progesterone. The intratissue conversion of progesterone to 5e-DHP (Table V) in the central gray was especially high whereas that of the anterior pituitary was low. DISCUSSION

The blood metabolic clearance rate of progesterone in the anesthetized female monkey was 295 ~ 45 (S.E.) 1/day. It appears that the MCR of progesterone in the anesthetized monkey (which is in the same range as the unanesthetized rhesus49), is somewhat lower than in women 2° based on their corresponding cardiac outputs 31. The head and brain extractions of progesterone were 30.4 -k 8.3 ~ (S.E.) and 26.0 ~: 9.1 °J,i (S.E.) or excluding the one high extraction (No. 7) 22.4 ± 2 . 7 ~ (S.E.) and 17.0 1.5 ~ (S.E.), respectively, which is similar to the sheep a. This unique measurement of the brain extraction (cf., head) was made possible by cannulation of the lateral sinus which in the monkey, unlike many other species, represents venous drainage only from the brain.

108 TABLE V MONKEY BRAIN DISTRIBUTION OF

[aH]5a-DHP AND TISSUECONVERSIONOF PROGESTERONETO 5a-DHP [aH]Sa-DHP AND [14C]PROGESTERONE

D U R I N G A C O N T I N U O U S INFUSION OF

~PSa TT is the intratissue transfer constant of progesterone to 5a-DHP and was calculated according to Chapdelaine7. Tissue

Anterior pituitary Amygdala Hippocampus Hypothalamus Remaining pituitary** Preoptic-anterior hypothalamus Cerebellum Central gray Spinal cord Cerebrum Thalamus Optic chiasm Medulla oblongata Pineal Pons Mesencephalic-tegmentum Pons reticular Fat

~":~Hj'5a-DHP

Ratio of / aHJSa-DHP

Ratio to cerebrum concentration

Ratio to carotid blood concentration

gesterone*

0.50 0.57 0.58 0.80 0.81 0.85 0.91 0.92 0.96 1.0 I. 1 1.1 1.2 1.4 1.4 1.6 1.6 0.28

1.8 2.0 2.0 2.9 2.9 3.0 3.2 3.3 3.4 3.5 4.1 3.9 4.3 4.9 4.9 5.5 5.8 1.0

0.49 0.77 0.91 1.1 1.3 1.1 1.1 1.8 0.82 1.1 1.0 0.92 1.0 1.2 1.1 1.0 1.2 1.1

~ TT~'~

to [14CJpro-

0.081 0.184 0.239 0.202 0.001 0.162 0.251 0.531 0.163 0.124 0.150 0.085 0.194 0.138 0.154 0.105 0.198 0.001

* Calculated from the ratio of [3H]5a-DHP and [14C]progesterone tissue concentrations to the [aH]5a-DHP and [!4C]progesterone carotid arterial blood concentrations. ** See legend of Table 1I.

F o r the m e a s u r e m e n t o f the rate o f tissue m e t a b o l i s m in the s t e a d y state by a r t e r i o v e n o u s c o n c e n t r a t i o n differences a c o n s t a n t b l o o d flow, a c o n s t a n t arterial conc e n t r a t i o n o f s u b s t r a t e a n d a c o n s t a n t rate o f tissue m e t a b o l i s m during the course o f s a m p l i n g m u s t be m a i n t a i n e d 54. W h i l e the latter two criteria a p p e a r to have been fulfilled in the p r e s e n t studies as i n d i c a t e d by coefficient o f v a r i a t i o n s o f the head e x t r a c t i o n o f p r o g e s t e r o n e o f 11 7O a n d o f b r a i n e x t r a c t i o n o f 15 ~ , we were unsuccessful in o u r a t t e m p t s to m e a s u r e the rate o f b l o o d flow. T h e b r a i n b l o o d flow o f the lightly anesthetized rhesus m o n k e y has been e s t i m a t e d to be a b o u t 50 ml/100 g / m i n ~7. The c a l c u l a t e d e s t i m a t e o f b r a i n clearance (extraction × b l o o d flow) o f p r o g e s t e r o n e w o u l d then be a b o u t 19 1/day o r 6 7o o f the m e t a b o l i c clearance rate. T h e r e a s o n for the high, b u t c o n s t a n t h e a d a n d b r a i n e x t r a c t i o n s o f p r o g e s t e r o n e in m o n k e y N o . 7 (Table I) is n o t k n o w n . Yates and U r q u h a r t 5~ h a v e suggested t h a t when the tissue e x t r a c t i o n o f a steroid is incomplete, the e x t r a c t i o n will b e c o m e m o r e d e p e n d e n t on the rate o f b l o o d flow. This a n i m a l m a y have h a d a lower c a r d i a c o u t p u t a n d b r a i n b l o o d flow.

109 Part of the head and brain extractions of progesterone can be attributed to the transhead and transbrain conversions of progesterone to 20a-OHP and 5a-DHP. The metabolism of progesterone to 20a-OHP and 5a-DHP would account for about 20 °.i~ of the head extraction and 30 ~ of the brain extraction. This compares with sheep in which the head conversion of progesterone to 20a-OHP accounts for 35 ~ of the progesterone extraction 3. It is not known what accounts for the remainder of the head and brain extractions of progesterone. To test whether our experimental protocol could be compared with other studies of the brain distribution of steroid hormones (e.g., in the ratlS), 1 monkey was given a continuous infusion of [3H]estradiol. The monkey's anterior pituitary had the highest concentration of [~H]estradiol and the hypothalamus had twice as much [3H]estradiol as the cerebrum (Table lIl), which is similar to what has been observed in rats by single injection and measurement of radioactivity (e.g., ref. 18) and in the monkey 13. In contrast to estradiol no area of the monkey brain or the pituitary contained a markedly higher concentration of progesterone than any other area although all areas had a higher concentration than did the carotid blood (Table Il). The brain distribution of progesterone in the female monkey was similar to that observed in ovariectomized guinea pigs16, 46, and the hind brain and midbrain contained higher concentrations of progesterone than the hypothalamus in both. The mesencephalon of the rat also concentrates more progesterone relative to other areas of the brain ~. It is not known what factors determine the regional brain distribution of progesterone although active metabolism of progesterone probably makes a major contribution to the relatively low levels of progesterone observed in the central gray (see below). High affinity, limited capacity receptor systems are thought to be largely responsible for the selective uptake and concentration of estradiol in the anterior pituitary and hypothalamusal,ls, 42 and perhaps other brain areas 2~. Attempts to identify 'receptor' molecules for progesterone or nuclear localization of progesterone in the rat or guinea pig anterior pituitary or hypothalamus by biochemical techniques have so far been mostly unsuccessful 1,9. By autoradiography nuclear binding of progesterone and/or its metabolites has been demonstrated in the nucleus arcuatus, the nucleus preopticus periventricularis and the nucleus preopticus suprachiasmaticus of the guinea pig hypothalamus 34 although another study has been negative (Discussion of ref. 1). The high lipid content of the brain might contribute to the brain distribution of progesterone and the similar brain distributions of 20a-OHP and 5aD H P (Tables IV and V) tends to support this suggestion 47. Insufficient brain sample size did not permit lipid analysis to test this hypothesis but adipose tissue from the abdomen in 3 out of 4 experiments had a much lower content of radioactive progesterone than did the brain tissues. A direct effect of progesterone on the pituitary of laboratory animals has been suggested on the basis of the response of the pituitary to extracts of the median eminence or to L H - R H 3~, but the monkey pituitary is as responsive to L H - R H during the luteal phase as during the follicular phase 12. The anterior pituitary had a higher concentration of radioactive progesterone than did the carotid blood (Table I l)

110 or the remainder of the pituitary although not significantly different from the cerebrum. The brain tissues could not only take up radioactive 20a-OHP from the circulation (Table IV) but could also actively convert progesterone to 20a-OHP, especially the cerebral cortex (Tables 1I and IV). The relatively low conversion of 20a-OHP to progesterone as indicated by the ~'a-T _20aP values (Table IV) is in agreement with the low brain extraction of 20a-OHP. Comparison of the head and brain extractions of 20aOHP (12.4 and 1.9~, respectively) illustrate the importance of sampling both the jugular vein and lateral sinus in studies of brain metabolism. The higher intratissue conversions of progesterone to 20a-OHP suggests that the 20a-OHP is being further metabolized in the brain before the radioactivity is released into the venous bloodL It is not known whether or not 20a-OHP can affect monkey brain function(s) or whether it is an inactive metabolite of progesterone. 20a-OHP does influence L H secretion in the rabbit 1~ and can also promote gonadotropin secretion in the estrogenprimed, ovariectomized raO 3. 20a-OHP also facilitates lordosis in the guinea pig ~ although it is relatively inactive in the rat zS. In contrast to 20a-OHP the brain extraction of 5a-DHP was appreciable and greater than that of progesterone, The progesterone-5a-DHP conversions (Table V) indicate that an appreciable portion of the 5a-DHP is formed from progesterone in the brain tissues themselves. Rat and baboon brain tissues can also convert progesterone to 5a-DHP in vitrola,17, 39. The central gray area has the highest conversion of progesterone to 5a-DHP (Table V) and the highest concentration of 5a-DHP formed from progesterone (Table I1). The mesencephalon and hind brain were more active than the cerebral cortex or anterior pituitary. Snipes and Shore 39 reported that the order of the in vitro conversion of progesterone to 5a-DHP by rat brain tissues was midbrain tegmentum ~ hypothalamus ~> cortex. It is not known if the conversion of progesterone to 5a-DHP in the monkey brain is an activation or inactivation mechanism of hormone action. 5a-DHP can facilitate ovulation in the immature rat treated with pregnant mare serum gonadotropin 41 and it also apparently reduces the basal release of L H and F S H by the male rat pituitary in vitro and inhibits the augmented release of gonadotropin in the response to L H - R H 36. 5a-DHP can also facilitate sexual receptivity in female rats 2~,5° and guinea pigs 4s. Although it is not as active as progesterone, 5a-DHP has a higher MCR than progesterone and its low solubility makes it a difficult substance to administer. The significance of the brain distribution of progesterone and its metabolites is not known. Although the distribution has been expressed here as a ratio to the cerebral cortex as a 'control', progesterone may also influence cortical functions (e.g., refs. 19, 33). Although regions of the hypothalamus have been implicated as sites for progesterone modulation of gonadotropin secretion and sex behavior ~6,z8,~0,34,38, implants of progesterone into the midbrain of rats facilitate lordosis a'z and in the guinea pig inhibit female sexual behavior 25. Stimulation or lesions of the central gray and certain other areas of the midbrain also affect gonadotropin reteasO ,~,s. The amygdala and hippocampus have also been suggested as areas which modulate gonadotropin secretion44,4L Recently, Piva et al. 29 have claimed that implants of

111 progesterone into the amygdala, cerebellum and median eminence of the castrated female rat will increase pituitary stores of L H and produce a decrease in the hypothalamic content of L H - R H . When one adds to these observations the fact that progesterone can also influence other brain functions such as sleep, aggression, appetite in some animals and basal body temperature, brain distribution studies will remain difficult to interpret until more definitive functions can be ascribed to specific neurons (e.g., ref. 22). The active metabolism of steroids by the central gray and the stimulation and lesion experiments of this area4, 6 suggest that more effort should be devoted to the central gray in neuroendocrinological studies of the biology of reproduction. ACKNOWLEDGEMENTS

We wish to acknowledge the excellent assistance of Remy Miguel and Augusts Heinsons in isolation of the steroids and Paul and Jim Austin in the animal experimentation. We also express our gratitude to Dr. L. Wolin for assistance in the anesthesia and Dr. W. Johnson for dissection of the brain samples of monkey No. 8. Appreciation is also extended to Mrs. K. Diamond for typing the manuscript. This study was supported by Grants from the U.S.P.H.S., HD-02378 and Program Project HD-07640. P.R. was a N.I.H. Training Fellow (HD-00024). R.B.B. was a recipient of a Career Development Award from the N.I.C.H.H.D., Grant HD42564.

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The metabolic clearance rate, head and brain extractions, and brain distribution and metabolism of progesterone in the anesthetized, female monkey (Macaca mulatta).

The brain distribution and metabolism of progesterone were studied in female, rhesus monkeys. Adult monkeys were anesthetized with ketamine and were g...
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