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Progesterone biotransformation by the pituitary and hypothalamus of rabbit
Journul of Srermd B,o,hrm,\rrr Prmtedu Great Br~ta,n
Vol
14. p,, 921 ,o 927. ,981
00224731
PROGESTERONE BIOTRANSFORMATION PITUITARY AND HYPOTHALAMUS OF RABBIT
81/090921-07102.00.0 Pergrmon Press Lid
BY THE
URMILA VERMA and KESHO R. LAUMAS
Department of Reproductive Biology. New Delhi-l
All India Institute 10029. India
of Medical
Sciences,
(Received 30 May 1980) SUMMARY Progesterone and its metabolites were localized in the neural tissues of rabbit against a blood concentration gradient when [‘HI-progesterone was infused at a constant rate (11 pCi/h) for 7-8 h. The concentration of C3H]-progesterone and its metabolites at the end of infusion varied between 80 and 125 mpCi/g in different parts of the brain. The endometrium and myometrium localized 130 and 55 mpCi/g tissue. respectively, while skeletal muscle concentrated only 6 m@/g tissue which was much lower than in plasma. 5x-Pregnane-3.20-dione was the major metabolite of progesterone in the hypothalamus while it was 2&x-hydroxypregn4en3-one in the pituitary. The metabolism of progesterone to 2&x-hydroxypregn4en-3-one. 5z-pregnane-3.20-dione and further reduction to 5a-pregnane-3/3-ol-20 one was much higher in the myometrial tissue than in the pituitary and hypothalamus. Of the subcellular fractions of hypothalamus, the nuclear preparation metabolized about 17.97; of progesterone, 5z-pregnane-3.2Odione being the major metabolite constituting 9.3% of the substrate. The maximal metabolism occurred in the soluble fraction where 2&z-hydroxypregn_Qen-3-one was the major metabolite constituting 10.6% of the substrate.
Radioactive
INTRODUCttON Progesterone has been shown to localize in the pituitary, hypothalamus and other brain parts and the reproductive tissues of rat [l-S]. The localization of progesterone in the brain centres and pituitary has been related to the sexual receptivity C&8]. The facilitatory and inhibitory actions of progesterone on the brain centres for the secretion of trophic hormones were demonstrated in rats [9. IO]. Moreover, negative feed-back effects of progesterone on the hypothalamic areas has been shown by the changes in the electrical activity and the behavioural patterns in rabbits [I I]. However, it is not clear whether these effects are brought about by progesterone alone or by its metabolites. The metabolism of progesterone was shown in the target organs [12-I53 and some of its metabolites have been suggested to have a biological role in pituitary and hypothalamus [lo, 16. 173. Therefore, in the present study to understand the metabolic fate of progesterone in rabbit pituitary and hypothalamus, biotransformation of progesterone in these tissues was investigated and compared with its metabolism in the uterus. MATERIALSAND METHODS Animals
Normal adult female rabbits weighing about 2.5 kg bred in the animal house of the All India Institute of Medical Sciences were used in this study. 921
steroids
For in vivu constant microinfusion studies [7-)H](SA 4.0Ci/mmol) and for in vitro biotransformation studies [1,2-3H]-progesterone (SA 47.8Ci/mmol) were used. The purity of the steroids was checked every two months by. t.1.c. using chloroform:acetone (9: 1. v/v). Purified [4-‘4C]-progesterone (SA SOmCi/mmol) was used for the determination of procedural losses. progesterone
lntruvenous infusion technique
Radiochemically pure [7-3H]-progesterone was dissolved in 0.5 ml ethanol and diluted with sterile 0.9% normal saline (9.5 ml) to give final concentration of 5% ethanol [18] and of about 0.7 pg of hormone per ml. The solution was infused intravenously through the ear vein with the help of IO ml glass syringe connected with a Teflon tubing to the rabbits, anaesthetized with sodium pentobarbitone (Nembutal Abbott India Ltd) which gives a stable anaesthesia [1921]. The infusion was maintained at a constant rate of 1.24 ml (11 @i)/h (0.87 pg,h) for 7-8 h with the aid of an infusion pump model No. 600-900 (Harvard Instrument Company, Massachusetts). At the end of infusion the animals were sacrificed, blood, uterus, pituitary and brain tissues were obtained immediately. Endometrium was scraped out from the myometrium. Hypothalamus, cerebral cortex, mid-brain, medulla oblongata, thalamus and cerebellum were separated carefully from the brain as described
URMILA VERMA
922
and
earlier [22]. The tissues were blotted to remove blood, minced and weighed immediately. Blood samples were centrifuged to separate plasma. The study was carried out with five different rabbits. Extraction
and fractionation
of progesterone
and
its
metabolites
The extraction of steroids and the preliminary fractionation of the extracts were carried out essentially as described by Lawson and Pearlman[18]. [C’“C]Progesterone (50 mCi/mmol) was used to estimate the procedural losses and corrections were made at each step of extraction and chromatography. Radioactive steroids from in uivo and in oitro studies were extracted thrice with acetone and once with methanol. The acetone and methanol extracts were combined and an aliquot was taken for the estimation of total extractable steroids. The steroid extract was concentrated under reduced pressure and diluted with 5 ml water. The aqueous extract was adjusted to pH 10 with 1 N NaOH and the free steroids were extracted thrice with 15 ml ether. The ether extracts were pooled and an aliquot was taken to estimate the free steroids. The aqueous phase was extracted twice with equal volume of n-butanol and an aliquot was taken to furnish the conjugated radio-metabolites. The ether extracts containing the free or non-conjugated radiometabolites were delipidized as described by Wiest[23], dried under nitrogen at 45’C and subjected to t.1.c. In vitro metabolism
of progesterone
For in citro metabolism studies, pituitary. hypothalamus and uterine tissues were pooled from three animals in each experiment. The results were obtained from the average of three experimental values. The tissues were obtained immediately after sacrificing the animals, blotted and placed on a clean petri dish on ice. The uterus was cut open longitudinally and the endometrium was gently scraped from the myometrium with the help of a sharp blade. The tissues were minced into small pieces and weighed on a Roller Smith torsion balance. The minced pituitary (40 mg), hypothalamus (60 mg) and myometrial tissues (about 500 mg) were incubated with [1,2-3H]-progesterone (40.8 pmol) for 2 h at 37C in Krebs-Ringer phosphate buffer [24] pH 7.4 supplemented with 1 mg glucose/ml. To study the metabolism of progesterone in subcellular fractions. hypothalamic tissue (100 mg) was pooled from 334 animals in each experiment and the results were expressed from the mean value of three experiments. The tissue was minced, homogenized with ice cold Krebs-Ringer phosphate buffer and fractionated by differential centrifugation to yield primary fractions of 8OOg nuclear, 10,000 g mitochondrial, 105,OOOg microsomal pellet and 105,OOOg cytosol preparations essentially according to Karavolas et al.[25]. The 8009 nuclear pellet was sus-
KESHO R. LAUMAS
pended in 2 ml of ice cold 0.01 M tris HCI buffer pH 7.4 containing 1 mM MgC12. 3 mM CaC12. 10mM KC1 and 0.25 M sucrose filtered through cheese cloth and centrifuged. The pellet was washed two more times with ice cold tris HCI buffer. Each wash was followed by centrifugation at 8009 for 10 min at 4 C. The nuclear. mitochondrial and microsomal pellets (suspended in KrebsRinger phosphate buffer) and the cytosol preparations were then incubated for 1 h with [1.2-3H]-progesterone (20.9 pmol) in the presence of 33 prnol of glucose, 11 pmol of glucose-6-phosphate, 2.3 pmol of ATP and 1.3 prnol of NADPH. The reaction was stopped by the addition of acetone. The steroids were extracted with acetone and methanol as described for in viuo studies. The free steroids were extracted with ether, delipidized and dried as described above and analysed for progesterone and its metabolites. Anulysis
From in vitro studies, the radioactive steroids were separated by t.1.c. on silica gel HF (0.25 mm thickness) plates using the following solvent systems: I. Hexaneethyl acetate (5:2, v/v), II. Chloroform-acetone (9: 1, v/v) III. Chloroform-methanol (99:1, v/v), IV. Hexane-diethyl ether (4: 1, v/v), V. Cyclohexene-cyclohexanone (9: 1, v/v) and paper chromatography using hexane-formamide solvent system as described earlier [14]. The steroids were located by ultraviolet light (254 nm) and by exposure to iodine vapours. Confirmation of the steroids was made by acetylation, saponification of the acetylated compounds, oxidation and recrystallization to constant specific activity as described earlier [26]. For in ciao studies progesterone and its metabolites separated by chromatography were pooled from five infusion experiments and used for identification studies. Radiouctiuity
counting
Radioactivity was measured in a liquid scintillation spectrometer at 58% counting efficiency (Packard Tricarb Model 3380) using 10ml of scintillation fluid (4 g of 2,5-diphenyloxazole and 0.1 g of 1,4&s-2(4methyl-5-phenyloxazolyl)-benzene dissolved in 1 1 of toluene. Quenching corrections were done using the internal standards. RESULTS
Uptake rabbit
of [7-jH)-progesterone
and its metabolites
in
tissues
The uptake of progesterone and its metabolites after a constant intravenous microinfusion of [7-jH]progesterone was significantly higher in different brain parts and uterine tissue than in plasma, while in the skeletal muscle it was still lower than in the plasma (Fig: 1). Localization of progesterone in various brain parts was not significantly different and it ranged from 80.9-125.2mpCi/g tissue. The uptake
Progesterone metabolism by rabbit pituitary and hypothalamus
923
Fig. 1. Uptake of [7-3H]-progesterone and its metabolites in terms of steroid concentration in rabbit tissues and plasma: The results are expressed as mpCi progesterone and its metabolites per g tissue and per ml plasma. The values are the mean + SEM of five sets of experiments.
(80.8 m&i/g)
in the cerebral cortex was comparatively lower than in the other brain parts investigated. In the endometrium progesterone localization (130.4m&i/g) was slightly higher than in different brain parts and about two and half times higher than in the myometrium (54.7 m$i/g). In the neural and uterine tissues the major amount of progesterone was present as ether extractable free steroids, while in the plasma, it was mainly in the form of conjugated steroids (Table 1) which revealed a rapid metabolism of progesterone in the blood. In vivo metabolism of progesterone The pattern of progesterone metabolism as seen in the ether extractable free fractions of these tissues revealed interesting differences (Table 2). Under in viva conditions Sa-pregnane-3,20-dione and Sa-pregnan-3/I-ol-20-one were formed much higher in the pituitary than in the hypothalamus. In the cerebral cortex 5a-pregnan-3/I-ol-20-one was found comparatively more than in the pituitary and hypothalamus. In the myometrium, both Sa-reduced metabolites and 20a-hydroxypregn-4-en-3-one were found in considerably high quantities. In plasma all these metabolites were found but the amount of most of the metabolites was comparatively lower than the tissues investigated. Major amount of the steroid accumulated in the plasma was in the form of highly polar metabolites of progesterone and only about 6% of the steroid was the unconverted progesterone.
In vitro metabolism of progesterone To rule out the plasma origin of these metabolites
in the tissues and to confirm the in situ formation of S.B.14,9--H
these metabolites, in vitro biotransformation studies were carried out. Ether extractable free steroids were analysed into progesterone and its metabolites. Figure 2 shows the per cent metabolites formed and per cent progesterone converted in pituitary, hypothalamus and myometrium. In the pituitary 2@x-hydroxypregn&en-3-one constituted to be the major metabolite being 3.1% of the total steroid while 5a-pregnane-3,20-dione and 5a-pregnan-3/?-ol-2O-one were comparatively less and together constituted to be 2.2%. In the hypothalamus, 5a-pregnane-3,20dione was the major metabolite (5%) formed. Formation of 5a-pregnane-3,20-dione and 5a-pregnan-3/Iol-20-one together constituted about 8% which was much higher than that of the formation of 20x-hydroxypregn-4-en-3-one (2.4”/,). The total amount of progesterone converted on unit weight basis by pituitary and hypothalamus was not significantly different. In the myometrial tissue progesterone was converted
Table 1. Percentage of the free and conjugated steroids in rabbit tissues and plasma Tissue Pituitary Hypothalamus Cerebral cortex Endometrium Myometrium Plasma
Free (ether extractable) steroids 67.6 89.0 94.0 77.9 81.2 32.5
k 2.6 & 2.0 k 1.8 * 5.4 &-3.1 f 1.9
Conjugated steroids 11.3 + 10.8 f 5.9 + 22.0 + 18.7 f 67.1 f
1.7 3.5 2.6 2.4 3.2 1.8
The values are the mean f SEM of five sets of experiments. The results are expressed as per cent free and conjugated steroids of the total extractable steroids.
URMILA VERMA and KESHO R. LAUMAS
924
Table 2. In ciao metabolism Sz-Pregnane3.20-dione
Tissue Pituitary Hypothalamus Cerebral cortex Myometrium Plasma
2.5 9.1 3.0 8.2 2.0
f t * k f
0.4 0.4 0.5 0.6 0.3
of progesterone
Progesterone converted 39.6 50.2 71.9 75.0 94.4
f f f + f
in rabbit
ZOa-Hydroxypregn-4-en-3-one
Sa-Pregnan-3B-ol20-one
1.2 2.1 2.1 I.9 4.6
3.1 10.1 II.1 15.6 4.3
* * k i *
tissues and plasma
0.2 0.5 0.6 0.3 0.3
8.3 3.0 2.5 6.6 2.3
i ) i + f
0.6 0.4 0.2 0.4 0.2
Polar compounds 20.3 27.3 55.3 44.3 76.2
+ + & + f
1.6 1.8 2.6 2.1 3.9
The results are expressed as per cent progesterone converted and per cent metabolites formed when ether extractable free steroids constituted IOO”,,. Constant intravenous microinfusion of [‘I-‘HI-progesterone and the extraction of the steroids was carried out as described under “Materials and Methods”. The ether extractable free steroids were analysed into progesterone and its metabolites. The results are an average + SEM of five sets of experiments.
to both 5a-pregnane-3,20-dione and 20cx-hydroxypregn-4-en-3-one almost equally. These in cirro metabolism studies showed that progesterone is biotransformed locally in these tissues. Conversion of progesterone to polar metabolites was much less under in cirro conditions as compared with in ciw conditions, where some of the polar metabolites may be diffused from blood. Suhcellulur
metabolism
qf
progesterone
in
hypothala-
mus The metabolism of progesterone in the nuclear, mitochondrial, microsomal and cytosolic fractions of the hypothalamus is shown in Table 3. With the nuclear fraction, on an average about 17.94, of progesterone was converted to its metabolites. 5x-Pregnane-3.20-dione was the major metabolite formed in this fraction. This constituted about 9.3”, of the substrate (Table 3). The pregnenolone isomer Sa-pregnan-3/I-ol-20-one was also formed in considerable amounts being 5.6% of the substrate. Very small amounts of 20a-hydroxypregn-4-en-3-one and the polar compounds were found in this fraction. With the mitochondrial preparation about 15% of progesterone was metabolised. k-pregnane-3,20-dione was the major metabolite of this fraction. Sa-pregnan-3fi-ol-20-one was also formed in considerably high amounts. Formation of polar compounds by this fraction as compared with other subcellular fractions was the highest. With the microsomal fraction about l2.8’(, of progesterone was metabolised and Sa-pregnan-3/?-ol-20-one was the major metabolite of this fraction. Metabolism of progesterone in the cytosol fraction was greater than with other subcellular fractions. On an average about 19.1’, of progesterone was metabolised. ZOr-hydroxypregn-4-en-3-one was the major metabolite which constituted about 10.6”,,, of the substrate. The formation of ring A saturated and polar metabolites was comparatively much lower. DISCUSSION
The present investigation revealed that in rabbit the metabolism of progesterone in the pituitary and
hypothalamus was qualitatively similar but there were interesting quantitative differences. See-Pregnane-3,20-dione was the prominent metabohte formed in the hypothalamus, while in the pituitary 2Oa-hydroxypregn-4-en-3-one was the major metabolite. _This suggested that Sa-reductase was more active in the hypothalamus while 20cr-hydroxysteroid dehydrogenase (20z-HSD) in the pituitary. On the other hand, in the myometrium these two metabolites were formed almost equally. Further reduction of 5u-pregnane-3,20-dione was much higher in the myometrium where more of 5a-pregnan-3/&ol-20-one was formed than in the pituitary and hypothalamus.
t
L PITUITARY
Fig. 2.
HYPOTHAIAMUS
metabolism of progesterone
MWXlETRIUL
in pituitary. hypothalamus and myometrial tissue: The results are expressed as per cent metabolites formed and as per cent progesterone converted when progesterone and its metabolites in ether extractable free steroids constituted lOO”,. The data is the mean f SEM of three sets of experiments. Pituitary (40 mg) hypothalamic tissue (60mn) and myometrial tissue (5UtJmg) pooled from three animals m each experiment were minced and incubated with [1,2-3H]-progesterone 41.9 pmol) at 37°C for 2 h. The steroids were extracted and analyzed as described under “Materials and Methods”. *5a-pregnane-3.20-dione q; 5a-pregnan-3pol-20-one IFI; progesterone a; 2&z-hydroxypregn-4-en-3one Cl: and polar compounds N. In
vitro
925
Progesterone metabolism by rabbit pituitary and hypothalamus Table 3. Metabolism of progesterone in the s&cellular fractions of rabbit hypothalamus Subcellular fractions
Steroids 5a-Pregnane-3,20-dione Progesterone converted Sa-Pregnan-3/J-01-20-one 20a-Hydroxypregn-4-en-3-one Polar compounds
Nuclear 8OOg 9.3 f 17.9 * 5.6 + 0.3 + 0.2 f
1.2 1.9 0.1 0.03 0.02
Mitochondrial 10,OOOg 6.6 f 15.0 f 4.9 f 1.0 f 2.3 *
0.9 1.1 0.9 0.02 0.1
Microsomal 105,000 g
Cytosol 105,OOOg Supernatant
4.0 f 12.8 + 4.4 f 1.9 * 1.3 f
2.5 + 19.1 + 2.8 k 10.6 f 1.4 f
1.1 1.2 0.7 0.1 0.1
0.4 1.1 0.2 0.5 0.1
The results are expressed as per cent progesterone converted and per cent metabolites formed. Hypothalamic tissue (100 mg) was fractionated into nuclear, mitochondrial, microsomal and cytosolic preparations. Each incubation was carried out in the presence of glucose 33 pmol, glucose-bphosphate 11 pmol, ATP 2.3 pmol, NADPH 1.3 pmol and [1,2-3H]-prozesterone 20.9 omol in Krebs Ringer ohosohate buffer (pH 7.4) at 37°C for 60 min. The values are mean + SEM of three sets of-experiments.
The cellular and subcellular metabolic pattern of progesterone in brain showed that Sa-reductase responsible for the formation of 5a-pregnane-3,20-dione was concentrated in the particulate fractions and mainly in the nuclear fraction. The nuclear localization of Sa-reductase has earlier been reported in the rat hypothalamus [27], rat cerebral cortex [28] and rat uterus [29]. 5a-Pregnane-3,20-dione is metabolised further to Sa-pregnan-3fi-ol-20-one in the particulate fractions. The conversion of progesterone to 20a-hydroxypregn-&en-3-one in the cytosol indicated the presence of the enzyme 20~HSD in this fraction. Further reduction of 5a-pregnan-3fi-ol-20-one and 20a-hydroxypregn-4-en-3-one leads to the formation of polar compounds. The cytosohc localization of the enzyme 20~HSD has been shown in the rabbit uterus [ 141. human uterus [ 15,301 and human skin [31]. These investigations on the metabolism of progesterone in the rabbit pituitary and hypothalamus have revealed the conversion of progesterone to its metabolites both under in viva and in vitro conditions. The Sa-reduced product of progesterone has been suggested to exert a positive feed-back effect in the dioestrus phase of the rat reproductive cycle for initiating oestrus [lo, 281. Recently Zanisi er a/.[173 have reported that, along with progesterone, Sa-pregnane-3-20-dione in the median eminence, causes a large increase of serum LH in rats implanted with ethinyl estradiol. Moreover, Martini[32] has reported that in castrated rats, progesterone does not inhibit either LH or FSH release in the absence of estrogen pre-treatment, while 5a-pregnane-3,20-dione exerts a strong blocking effect on both gonadotrophins. 5a-Pregnan-3/Gol-20-one has also been suggested to play a physiological role in the process leading to gonadotrophin release [33]. The formation of significant amounts of 2Ou-hydroxypregr&en-3-one in the pituitary may bear a special significance, since it has been reported to act as a positive feed-back agent to prolong and heighten LH discharge in the mated rabbits [ 171, and in rats it has synergistic effect with oes-
tradiol-17/? in the induction of prooestrus LH surge [34,35]. It is possible that the varied central nervous system effects are due to the combined action of progresterone and some of its metabolic products in varying concentrations under different physiological conditions. This may be brought about by the binding of progesterone and some of its metabolites to the specific receptors in pituitary and hypothalamus. Although progesterone receptors have been well demonstrated in the reproductive organs [36381, conflicting data existed on progesterone receptors in brain. Atger et aI.[39] and Kato[40] failed in their earlier studies to demonstrate the presence of specific progesterone receptors in pituitary and hypothalamus while under identical conditons specific progesterone receptors were present in uterus. However, later studies have shown the presence of specific progesterone receptors in brain tissues [4145]. Further studies at the molecular level would demonstrate the direct action of progesterone and its metabolites on the hypothalamo-pituitary axis. Acknowledgement-This work was supported by the grants from the Ford Foundation, New York and the World Health Organization, Geneva.
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926
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metabolism of progesterone 23. Wiest W. G.: Extrahepatic in pseudopregnant rats. Identification of reduction products. J. biol. Chrm. 238 (1963) 94-99. and study of 24. Cohen P. P.: Methods for preparation tissues. In .~u~zf~rnefri~, ~ec/~~j~~e.s (Edited by Umbreit W. W., Burnis R. H. and StaulTer T. F.). Burges., Minneapolis (1959) pp. 135-169. 25. Karavolas H. J.. Meyer R. K.. Deighton K. 5. and Adrouny S: Stimulation and inhibition of synthesis of ovulating hormone (OH) in organ culture by subcellular fractions of rat medial basal hypothalami. Endocri~~~~~g~ 88 ( 197 i ) 969-975. K., Hingorani V. and Laumas K. R.: 1n 26. Murugesan vitro metabolism of norethynodrel in the human endometrium and myometrium. A&r Endocr.. Copenh. 74 (1973) 576-591. 27. Karavolas H. J. and Herf S. M.: Conversion of progesterone by rat medial basal hypothalamic tissue to Sz-pregnane-3,20-dione. ~~d~~crj~z~/og~ 89 (1971) 940-942. 28. Tabei T.. Haga H.. Heinrichs W. L. R. and Herrmann W. L.: Metabolism of progesterone bv rat brain. nituitary gland and other Tissues. St&ids 23 (‘1974) 651-666. 29. Wichman. K.: On the metabolism and subcellular distribution of progesterone in the myometrium of the pregnant rat. Acttr Endocr., Cop&. Suppl. 116 (1967) t-98. M. D.. Bridges C. E. and 30. Collins W. P.. Mansfield Sommerville 1. F.: Studies on steroid metabolism in human endometrial tissue. Bj~~~7~~m. J. 113 (1969) 399-407. 31. Gomez E. C., Frost M. D. and Hsia, S. L.: Transformation of progesterone by subcellular fractions of human skin. Incest Der~uro~. 64 (1975) 240-244. of progesterone in the brain 32. Martini L.: Metabolism and in the anterior pituitary: significance for the control of gonadotropin secretion. Acttr Biol. A&. Sci. Hf~~g. 28 (1977) I 9. 33. Zanisi M. and Martini L.: Effects of progesterone metabolites on gonadotrophin secretion. J. steroid Bioehrm. 6 (1975) 1021-1023. 34. Swerdloff R. S.. Jacobs H. S. and Ode11 W. D.: Synergistic role of progestogens in estrogen induction of LH and FSH surge. Endocrino/ogj, 90 (1972) 1529. 1536. 35. Martini L. Celotti F., Juneja H.. Motta M. and Zanisi M.: Feedback effects on central mechanisms controiling neuroendocrine functions. In Central Regulurion 01 rhe Endocrine System (Edited by Juxe. K., Hokfelt 0. and Luft R.). Plenum Publishing Corporation (1979) pp. 273-295. in uterus 36. Milgrom E. and Baulieu E. E.: Progesterone and plasma. I. Binding in rat uterus 105.000 x g supernatant. Endocrinofog~ 87 (1970) 276-287. B. W.. McGuire W. L.. Kohler P. 0. and 37. O’Malley Korenman S. Cr.: Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. In Recent Proyress iI1 Hormone Reseurch (Edited by Astwood E. B.). Academic Press. New York. Vol. 25 (t969) pp. 105-160. 38. Verma U. and Laumas K. R.: Itt rifro binding of progesterone to receptors in the human endometrium and the myometrium. Bioc~him. hioph!x Autr 317 (1973) 403-419.
39. Atger M.. Baulieu E. E. and Milgrom E.: An investigation of progesterone receptors in guinea pia vagina. uterine cervix. mammary gland, piiuitary, ‘hipothalamus. ~~d~~cri~~~~u~ 94 (1974) 161- 167. and hypophyseal 40. Kato Z.: The roie of hypothalamic 5x-dihydrotestosterone. estradiol and progesterone receptors in the mechanism of feedback action. J. sreroid Biochem. 6 (1975) 979-987.
Progesterone metabolism by rabbit pituitary and hypothalamus 41. Roy J. E., Maclusky N. J. and McEwen B. S.: Antiestrogen inhibits the induction of progestin receptors by estradiol in the hypothalamus preoptic area and pituitary. Endocrinologp 104 (1979) 1333-t 336. 42. Kato J. and Onouchi T.: Nuclear progesterone receptors and characterization of cytosol receptors in the rat hypothalamus and anterior hypophysis. J. steroid Biothem. 11 (1979) 845-854. 43. MacLusky N. J., Liebergurg I., Krey L. S. and McEwen B. S.: Progestin receptors in the brain and pituitary of the bonnet monkey (Macaca radiata): Dif-
927
ferences between the monkey and the rat in the distribution of progestin receptors. Endocrinology 106 (1980) 185-191. 44. MacLusky N. J. and McEwen B. S.: Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinoloy~ 106 (1980) 192-202. 45. Blaustein J. D. and Feder H. H.: Nuclear progestin receptors in guinea pig brain measured by an in rirro exchange assay after hormonal treatments that affect lordosis. Endocrinology 106 (1980) 1061-1069.
Vol
14. p,, 921 ,o 927. ,981
00224731
PROGESTERONE BIOTRANSFORMATION PITUITARY AND HYPOTHALAMUS OF RABBIT
81/090921-07102.00.0 Pergrmon Press Lid
BY THE
URMILA VERMA and KESHO R. LAUMAS
Department of Reproductive Biology. New Delhi-l
All India Institute 10029. India
of Medical
Sciences,
(Received 30 May 1980) SUMMARY Progesterone and its metabolites were localized in the neural tissues of rabbit against a blood concentration gradient when [‘HI-progesterone was infused at a constant rate (11 pCi/h) for 7-8 h. The concentration of C3H]-progesterone and its metabolites at the end of infusion varied between 80 and 125 mpCi/g in different parts of the brain. The endometrium and myometrium localized 130 and 55 mpCi/g tissue. respectively, while skeletal muscle concentrated only 6 m@/g tissue which was much lower than in plasma. 5x-Pregnane-3.20-dione was the major metabolite of progesterone in the hypothalamus while it was 2&x-hydroxypregn4en3-one in the pituitary. The metabolism of progesterone to 2&x-hydroxypregn4en-3-one. 5z-pregnane-3.20-dione and further reduction to 5a-pregnane-3/3-ol-20 one was much higher in the myometrial tissue than in the pituitary and hypothalamus. Of the subcellular fractions of hypothalamus, the nuclear preparation metabolized about 17.97; of progesterone, 5z-pregnane-3.2Odione being the major metabolite constituting 9.3% of the substrate. The maximal metabolism occurred in the soluble fraction where 2&z-hydroxypregn_Qen-3-one was the major metabolite constituting 10.6% of the substrate.
Radioactive
INTRODUCttON Progesterone has been shown to localize in the pituitary, hypothalamus and other brain parts and the reproductive tissues of rat [l-S]. The localization of progesterone in the brain centres and pituitary has been related to the sexual receptivity C&8]. The facilitatory and inhibitory actions of progesterone on the brain centres for the secretion of trophic hormones were demonstrated in rats [9. IO]. Moreover, negative feed-back effects of progesterone on the hypothalamic areas has been shown by the changes in the electrical activity and the behavioural patterns in rabbits [I I]. However, it is not clear whether these effects are brought about by progesterone alone or by its metabolites. The metabolism of progesterone was shown in the target organs [12-I53 and some of its metabolites have been suggested to have a biological role in pituitary and hypothalamus [lo, 16. 173. Therefore, in the present study to understand the metabolic fate of progesterone in rabbit pituitary and hypothalamus, biotransformation of progesterone in these tissues was investigated and compared with its metabolism in the uterus. MATERIALSAND METHODS Animals
Normal adult female rabbits weighing about 2.5 kg bred in the animal house of the All India Institute of Medical Sciences were used in this study. 921
steroids
For in vivu constant microinfusion studies [7-)H](SA 4.0Ci/mmol) and for in vitro biotransformation studies [1,2-3H]-progesterone (SA 47.8Ci/mmol) were used. The purity of the steroids was checked every two months by. t.1.c. using chloroform:acetone (9: 1. v/v). Purified [4-‘4C]-progesterone (SA SOmCi/mmol) was used for the determination of procedural losses. progesterone
lntruvenous infusion technique
Radiochemically pure [7-3H]-progesterone was dissolved in 0.5 ml ethanol and diluted with sterile 0.9% normal saline (9.5 ml) to give final concentration of 5% ethanol [18] and of about 0.7 pg of hormone per ml. The solution was infused intravenously through the ear vein with the help of IO ml glass syringe connected with a Teflon tubing to the rabbits, anaesthetized with sodium pentobarbitone (Nembutal Abbott India Ltd) which gives a stable anaesthesia [1921]. The infusion was maintained at a constant rate of 1.24 ml (11 @i)/h (0.87 pg,h) for 7-8 h with the aid of an infusion pump model No. 600-900 (Harvard Instrument Company, Massachusetts). At the end of infusion the animals were sacrificed, blood, uterus, pituitary and brain tissues were obtained immediately. Endometrium was scraped out from the myometrium. Hypothalamus, cerebral cortex, mid-brain, medulla oblongata, thalamus and cerebellum were separated carefully from the brain as described
URMILA VERMA
922
and
earlier [22]. The tissues were blotted to remove blood, minced and weighed immediately. Blood samples were centrifuged to separate plasma. The study was carried out with five different rabbits. Extraction
and fractionation
of progesterone
and
its
metabolites
The extraction of steroids and the preliminary fractionation of the extracts were carried out essentially as described by Lawson and Pearlman[18]. [C’“C]Progesterone (50 mCi/mmol) was used to estimate the procedural losses and corrections were made at each step of extraction and chromatography. Radioactive steroids from in uivo and in oitro studies were extracted thrice with acetone and once with methanol. The acetone and methanol extracts were combined and an aliquot was taken for the estimation of total extractable steroids. The steroid extract was concentrated under reduced pressure and diluted with 5 ml water. The aqueous extract was adjusted to pH 10 with 1 N NaOH and the free steroids were extracted thrice with 15 ml ether. The ether extracts were pooled and an aliquot was taken to estimate the free steroids. The aqueous phase was extracted twice with equal volume of n-butanol and an aliquot was taken to furnish the conjugated radio-metabolites. The ether extracts containing the free or non-conjugated radiometabolites were delipidized as described by Wiest[23], dried under nitrogen at 45’C and subjected to t.1.c. In vitro metabolism
of progesterone
For in citro metabolism studies, pituitary. hypothalamus and uterine tissues were pooled from three animals in each experiment. The results were obtained from the average of three experimental values. The tissues were obtained immediately after sacrificing the animals, blotted and placed on a clean petri dish on ice. The uterus was cut open longitudinally and the endometrium was gently scraped from the myometrium with the help of a sharp blade. The tissues were minced into small pieces and weighed on a Roller Smith torsion balance. The minced pituitary (40 mg), hypothalamus (60 mg) and myometrial tissues (about 500 mg) were incubated with [1,2-3H]-progesterone (40.8 pmol) for 2 h at 37C in Krebs-Ringer phosphate buffer [24] pH 7.4 supplemented with 1 mg glucose/ml. To study the metabolism of progesterone in subcellular fractions. hypothalamic tissue (100 mg) was pooled from 334 animals in each experiment and the results were expressed from the mean value of three experiments. The tissue was minced, homogenized with ice cold Krebs-Ringer phosphate buffer and fractionated by differential centrifugation to yield primary fractions of 8OOg nuclear, 10,000 g mitochondrial, 105,OOOg microsomal pellet and 105,OOOg cytosol preparations essentially according to Karavolas et al.[25]. The 8009 nuclear pellet was sus-
KESHO R. LAUMAS
pended in 2 ml of ice cold 0.01 M tris HCI buffer pH 7.4 containing 1 mM MgC12. 3 mM CaC12. 10mM KC1 and 0.25 M sucrose filtered through cheese cloth and centrifuged. The pellet was washed two more times with ice cold tris HCI buffer. Each wash was followed by centrifugation at 8009 for 10 min at 4 C. The nuclear. mitochondrial and microsomal pellets (suspended in KrebsRinger phosphate buffer) and the cytosol preparations were then incubated for 1 h with [1.2-3H]-progesterone (20.9 pmol) in the presence of 33 prnol of glucose, 11 pmol of glucose-6-phosphate, 2.3 pmol of ATP and 1.3 prnol of NADPH. The reaction was stopped by the addition of acetone. The steroids were extracted with acetone and methanol as described for in viuo studies. The free steroids were extracted with ether, delipidized and dried as described above and analysed for progesterone and its metabolites. Anulysis
From in vitro studies, the radioactive steroids were separated by t.1.c. on silica gel HF (0.25 mm thickness) plates using the following solvent systems: I. Hexaneethyl acetate (5:2, v/v), II. Chloroform-acetone (9: 1, v/v) III. Chloroform-methanol (99:1, v/v), IV. Hexane-diethyl ether (4: 1, v/v), V. Cyclohexene-cyclohexanone (9: 1, v/v) and paper chromatography using hexane-formamide solvent system as described earlier [14]. The steroids were located by ultraviolet light (254 nm) and by exposure to iodine vapours. Confirmation of the steroids was made by acetylation, saponification of the acetylated compounds, oxidation and recrystallization to constant specific activity as described earlier [26]. For in ciao studies progesterone and its metabolites separated by chromatography were pooled from five infusion experiments and used for identification studies. Radiouctiuity
counting
Radioactivity was measured in a liquid scintillation spectrometer at 58% counting efficiency (Packard Tricarb Model 3380) using 10ml of scintillation fluid (4 g of 2,5-diphenyloxazole and 0.1 g of 1,4&s-2(4methyl-5-phenyloxazolyl)-benzene dissolved in 1 1 of toluene. Quenching corrections were done using the internal standards. RESULTS
Uptake rabbit
of [7-jH)-progesterone
and its metabolites
in
tissues
The uptake of progesterone and its metabolites after a constant intravenous microinfusion of [7-jH]progesterone was significantly higher in different brain parts and uterine tissue than in plasma, while in the skeletal muscle it was still lower than in the plasma (Fig: 1). Localization of progesterone in various brain parts was not significantly different and it ranged from 80.9-125.2mpCi/g tissue. The uptake
Progesterone metabolism by rabbit pituitary and hypothalamus
923
Fig. 1. Uptake of [7-3H]-progesterone and its metabolites in terms of steroid concentration in rabbit tissues and plasma: The results are expressed as mpCi progesterone and its metabolites per g tissue and per ml plasma. The values are the mean + SEM of five sets of experiments.
(80.8 m&i/g)
in the cerebral cortex was comparatively lower than in the other brain parts investigated. In the endometrium progesterone localization (130.4m&i/g) was slightly higher than in different brain parts and about two and half times higher than in the myometrium (54.7 m$i/g). In the neural and uterine tissues the major amount of progesterone was present as ether extractable free steroids, while in the plasma, it was mainly in the form of conjugated steroids (Table 1) which revealed a rapid metabolism of progesterone in the blood. In vivo metabolism of progesterone The pattern of progesterone metabolism as seen in the ether extractable free fractions of these tissues revealed interesting differences (Table 2). Under in viva conditions Sa-pregnane-3,20-dione and Sa-pregnan-3/I-ol-20-one were formed much higher in the pituitary than in the hypothalamus. In the cerebral cortex 5a-pregnan-3/I-ol-20-one was found comparatively more than in the pituitary and hypothalamus. In the myometrium, both Sa-reduced metabolites and 20a-hydroxypregn-4-en-3-one were found in considerably high quantities. In plasma all these metabolites were found but the amount of most of the metabolites was comparatively lower than the tissues investigated. Major amount of the steroid accumulated in the plasma was in the form of highly polar metabolites of progesterone and only about 6% of the steroid was the unconverted progesterone.
In vitro metabolism of progesterone To rule out the plasma origin of these metabolites
in the tissues and to confirm the in situ formation of S.B.14,9--H
these metabolites, in vitro biotransformation studies were carried out. Ether extractable free steroids were analysed into progesterone and its metabolites. Figure 2 shows the per cent metabolites formed and per cent progesterone converted in pituitary, hypothalamus and myometrium. In the pituitary 2@x-hydroxypregn&en-3-one constituted to be the major metabolite being 3.1% of the total steroid while 5a-pregnane-3,20-dione and 5a-pregnan-3/?-ol-2O-one were comparatively less and together constituted to be 2.2%. In the hypothalamus, 5a-pregnane-3,20dione was the major metabolite (5%) formed. Formation of 5a-pregnane-3,20-dione and 5a-pregnan-3/Iol-20-one together constituted about 8% which was much higher than that of the formation of 20x-hydroxypregn-4-en-3-one (2.4”/,). The total amount of progesterone converted on unit weight basis by pituitary and hypothalamus was not significantly different. In the myometrial tissue progesterone was converted
Table 1. Percentage of the free and conjugated steroids in rabbit tissues and plasma Tissue Pituitary Hypothalamus Cerebral cortex Endometrium Myometrium Plasma
Free (ether extractable) steroids 67.6 89.0 94.0 77.9 81.2 32.5
k 2.6 & 2.0 k 1.8 * 5.4 &-3.1 f 1.9
Conjugated steroids 11.3 + 10.8 f 5.9 + 22.0 + 18.7 f 67.1 f
1.7 3.5 2.6 2.4 3.2 1.8
The values are the mean f SEM of five sets of experiments. The results are expressed as per cent free and conjugated steroids of the total extractable steroids.
URMILA VERMA and KESHO R. LAUMAS
924
Table 2. In ciao metabolism Sz-Pregnane3.20-dione
Tissue Pituitary Hypothalamus Cerebral cortex Myometrium Plasma
2.5 9.1 3.0 8.2 2.0
f t * k f
0.4 0.4 0.5 0.6 0.3
of progesterone
Progesterone converted 39.6 50.2 71.9 75.0 94.4
f f f + f
in rabbit
ZOa-Hydroxypregn-4-en-3-one
Sa-Pregnan-3B-ol20-one
1.2 2.1 2.1 I.9 4.6
3.1 10.1 II.1 15.6 4.3
* * k i *
tissues and plasma
0.2 0.5 0.6 0.3 0.3
8.3 3.0 2.5 6.6 2.3
i ) i + f
0.6 0.4 0.2 0.4 0.2
Polar compounds 20.3 27.3 55.3 44.3 76.2
+ + & + f
1.6 1.8 2.6 2.1 3.9
The results are expressed as per cent progesterone converted and per cent metabolites formed when ether extractable free steroids constituted IOO”,,. Constant intravenous microinfusion of [‘I-‘HI-progesterone and the extraction of the steroids was carried out as described under “Materials and Methods”. The ether extractable free steroids were analysed into progesterone and its metabolites. The results are an average + SEM of five sets of experiments.
to both 5a-pregnane-3,20-dione and 20cx-hydroxypregn-4-en-3-one almost equally. These in cirro metabolism studies showed that progesterone is biotransformed locally in these tissues. Conversion of progesterone to polar metabolites was much less under in cirro conditions as compared with in ciw conditions, where some of the polar metabolites may be diffused from blood. Suhcellulur
metabolism
qf
progesterone
in
hypothala-
mus The metabolism of progesterone in the nuclear, mitochondrial, microsomal and cytosolic fractions of the hypothalamus is shown in Table 3. With the nuclear fraction, on an average about 17.94, of progesterone was converted to its metabolites. 5x-Pregnane-3.20-dione was the major metabolite formed in this fraction. This constituted about 9.3”, of the substrate (Table 3). The pregnenolone isomer Sa-pregnan-3/I-ol-20-one was also formed in considerable amounts being 5.6% of the substrate. Very small amounts of 20a-hydroxypregn-4-en-3-one and the polar compounds were found in this fraction. With the mitochondrial preparation about 15% of progesterone was metabolised. k-pregnane-3,20-dione was the major metabolite of this fraction. Sa-pregnan-3fi-ol-20-one was also formed in considerably high amounts. Formation of polar compounds by this fraction as compared with other subcellular fractions was the highest. With the microsomal fraction about l2.8’(, of progesterone was metabolised and Sa-pregnan-3/?-ol-20-one was the major metabolite of this fraction. Metabolism of progesterone in the cytosol fraction was greater than with other subcellular fractions. On an average about 19.1’, of progesterone was metabolised. ZOr-hydroxypregn-4-en-3-one was the major metabolite which constituted about 10.6”,,, of the substrate. The formation of ring A saturated and polar metabolites was comparatively much lower. DISCUSSION
The present investigation revealed that in rabbit the metabolism of progesterone in the pituitary and
hypothalamus was qualitatively similar but there were interesting quantitative differences. See-Pregnane-3,20-dione was the prominent metabohte formed in the hypothalamus, while in the pituitary 2Oa-hydroxypregn-4-en-3-one was the major metabolite. _This suggested that Sa-reductase was more active in the hypothalamus while 20cr-hydroxysteroid dehydrogenase (20z-HSD) in the pituitary. On the other hand, in the myometrium these two metabolites were formed almost equally. Further reduction of 5u-pregnane-3,20-dione was much higher in the myometrium where more of 5a-pregnan-3/&ol-20-one was formed than in the pituitary and hypothalamus.
t
L PITUITARY
Fig. 2.
HYPOTHAIAMUS
metabolism of progesterone
MWXlETRIUL
in pituitary. hypothalamus and myometrial tissue: The results are expressed as per cent metabolites formed and as per cent progesterone converted when progesterone and its metabolites in ether extractable free steroids constituted lOO”,. The data is the mean f SEM of three sets of experiments. Pituitary (40 mg) hypothalamic tissue (60mn) and myometrial tissue (5UtJmg) pooled from three animals m each experiment were minced and incubated with [1,2-3H]-progesterone 41.9 pmol) at 37°C for 2 h. The steroids were extracted and analyzed as described under “Materials and Methods”. *5a-pregnane-3.20-dione q; 5a-pregnan-3pol-20-one IFI; progesterone a; 2&z-hydroxypregn-4-en-3one Cl: and polar compounds N. In
vitro
925
Progesterone metabolism by rabbit pituitary and hypothalamus Table 3. Metabolism of progesterone in the s&cellular fractions of rabbit hypothalamus Subcellular fractions
Steroids 5a-Pregnane-3,20-dione Progesterone converted Sa-Pregnan-3/J-01-20-one 20a-Hydroxypregn-4-en-3-one Polar compounds
Nuclear 8OOg 9.3 f 17.9 * 5.6 + 0.3 + 0.2 f
1.2 1.9 0.1 0.03 0.02
Mitochondrial 10,OOOg 6.6 f 15.0 f 4.9 f 1.0 f 2.3 *
0.9 1.1 0.9 0.02 0.1
Microsomal 105,000 g
Cytosol 105,OOOg Supernatant
4.0 f 12.8 + 4.4 f 1.9 * 1.3 f
2.5 + 19.1 + 2.8 k 10.6 f 1.4 f
1.1 1.2 0.7 0.1 0.1
0.4 1.1 0.2 0.5 0.1
The results are expressed as per cent progesterone converted and per cent metabolites formed. Hypothalamic tissue (100 mg) was fractionated into nuclear, mitochondrial, microsomal and cytosolic preparations. Each incubation was carried out in the presence of glucose 33 pmol, glucose-bphosphate 11 pmol, ATP 2.3 pmol, NADPH 1.3 pmol and [1,2-3H]-prozesterone 20.9 omol in Krebs Ringer ohosohate buffer (pH 7.4) at 37°C for 60 min. The values are mean + SEM of three sets of-experiments.
The cellular and subcellular metabolic pattern of progesterone in brain showed that Sa-reductase responsible for the formation of 5a-pregnane-3,20-dione was concentrated in the particulate fractions and mainly in the nuclear fraction. The nuclear localization of Sa-reductase has earlier been reported in the rat hypothalamus [27], rat cerebral cortex [28] and rat uterus [29]. 5a-Pregnane-3,20-dione is metabolised further to Sa-pregnan-3fi-ol-20-one in the particulate fractions. The conversion of progesterone to 20a-hydroxypregn-&en-3-one in the cytosol indicated the presence of the enzyme 20~HSD in this fraction. Further reduction of 5a-pregnan-3fi-ol-20-one and 20a-hydroxypregn-4-en-3-one leads to the formation of polar compounds. The cytosohc localization of the enzyme 20~HSD has been shown in the rabbit uterus [ 141. human uterus [ 15,301 and human skin [31]. These investigations on the metabolism of progesterone in the rabbit pituitary and hypothalamus have revealed the conversion of progesterone to its metabolites both under in viva and in vitro conditions. The Sa-reduced product of progesterone has been suggested to exert a positive feed-back effect in the dioestrus phase of the rat reproductive cycle for initiating oestrus [lo, 281. Recently Zanisi er a/.[173 have reported that, along with progesterone, Sa-pregnane-3-20-dione in the median eminence, causes a large increase of serum LH in rats implanted with ethinyl estradiol. Moreover, Martini[32] has reported that in castrated rats, progesterone does not inhibit either LH or FSH release in the absence of estrogen pre-treatment, while 5a-pregnane-3,20-dione exerts a strong blocking effect on both gonadotrophins. 5a-Pregnan-3/Gol-20-one has also been suggested to play a physiological role in the process leading to gonadotrophin release [33]. The formation of significant amounts of 2Ou-hydroxypregr&en-3-one in the pituitary may bear a special significance, since it has been reported to act as a positive feed-back agent to prolong and heighten LH discharge in the mated rabbits [ 171, and in rats it has synergistic effect with oes-
tradiol-17/? in the induction of prooestrus LH surge [34,35]. It is possible that the varied central nervous system effects are due to the combined action of progresterone and some of its metabolic products in varying concentrations under different physiological conditions. This may be brought about by the binding of progesterone and some of its metabolites to the specific receptors in pituitary and hypothalamus. Although progesterone receptors have been well demonstrated in the reproductive organs [36381, conflicting data existed on progesterone receptors in brain. Atger et aI.[39] and Kato[40] failed in their earlier studies to demonstrate the presence of specific progesterone receptors in pituitary and hypothalamus while under identical conditons specific progesterone receptors were present in uterus. However, later studies have shown the presence of specific progesterone receptors in brain tissues [4145]. Further studies at the molecular level would demonstrate the direct action of progesterone and its metabolites on the hypothalamo-pituitary axis. Acknowledgement-This work was supported by the grants from the Ford Foundation, New York and the World Health Organization, Geneva.
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URMILA VERMAand KESHOR. LA~JMAS
926
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