Target cells for dihydrotestosterone in the guinea pig pituitary gland

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Brain Research, 362 (1986) 318-321 Elsev,er

BRE 11353

Target Cells for Dihydrotestosterone in the Guinea Pig Pituitary Gland DAMON C HERBERT and PETER J. SHERIDAN Department of Cellular and Structural Btology, Dtvtston of Reproducttve Bzology, The Untverslty of Texas Health Sctence Center, San Antomo, TX78284 (U.S A ) (Accepted May 7th, 1985) Key words guinea pig - - autora&ography - - lmmunocytochemistry - - dihydrotestosterone - - gonadotroph - thyrotroph-pituicyte

Adult male guinea pigs were castrated and 24 h later injected with [3H]dlhydrotestosterone The,r p~tmtary glands were then processed for autoradiographlc and immunocytochemical analyses Three percent of the parenchymal cells in the pars distalls were radiolabelled. These cells were primanly gonadotrophs; a small populat,on of thyrotrophs were also found to retain the [3H]androgen Labelling occurred m cells of the pars intermedla (3 2%) as well as in 9 6% of the pltmcytes. These data m&cate that in addition to gonadotrophs, the pars nervosa is a major target for &hydrotestosterone m the guinea pig pituitary gland INTRODUCTION The pituitary gland has long been known to be a site for the feedback of the gonadal steroid h o r m o n e s progesterone, estrogen and testosterone. The role of these h o r m o n e s m the regulation of g o n a d o t r o p h i n secretion is well established. The technique of autoradiography has been e m p l o y e d to identify the location of target cells for the steroid h o r m o n e s within the hypophysls of a n u m b e r of animal speciesl8,19. In general, the main region of the pituitary gland which binds estrogen and p r o g e s t e r o n e is the pars distalis5,7,10,13,23 A similar finding is not always noted, however, with either testosterone or dihydrotestosterone ( D H T ) . In some species, a m a j o r i t y of the binding will be seen in the cells of the pars distalis6,14,17, whde m others, such as in the b a b o o n 4 and mouse 16, the pitmcytes in the pars nervosa comprise the m a j o r cell population which retains [3H]DHT. Considering the lack of unammlty with regard to the prime target cells for the androgens, we decided to study the uptake of tritiated D H T m the pitmtary gland of a commonly s t u & e d l a b o r a t o r y animal, the guinea pig A portion of our work also e m p l o y e d immunocytochemistry in conJunction with the autora-

dlography so as to p r e o s e l y identify the steroid-sensitive cells within the pars distalis. MATERIALS AND METHODS Six adult male guinea pigs ranging m weight from 526 to 603 g were gonadectomized. Twenty-four hours later, each animal was anesthetized with sodium pentabarbital and given an intravenous injection of 0.2/~g/100 g body weight 5a-dihydro-[1,2,4,5,6,73H]testosterone. O n e of the animals was designated as a control and received, in addition to the tritiated steroid, 20/~g/100 g body weight unlabelled D H T . The radiolabelled steroid was purchased from A m e r s h a m and had a specific activity of 147 Cl/mmol One-and-a-half hours after receiving the [3H]DHT, the animals were decapitated. The pituitary glands were rapidly r e m o v e d , m o u n t e d on brass tissue holders covered with minced liver and immersed m liquified propane. F o u r m i c r o m e t e r thick frozen (-35 °C) sections were o b t a i n e d usmg a Harris Wide Range Cryostat. The sections were placed on shdes coated with K o d a k NTB-2 emulsion and stored for 13-24 months at - 1 5 °C. The slides were then etther developed and stained with hematoxyhn

Correspondence D C Herbert, Department of Cellular and Structural Biology, The Umverslty of Texas Health Soence Center, San Antomo, TX 78284, U S A 0006-8993/86/$03 50 © 1986 Elsev,er Science Pubhshers B V (Biomedical Division)

319 and eosin 8 or fixed developed and immunocytochemically stained n using antibodies to the beta subunits of ovine luteinizing h o r m o n e (LHfl) or of human thyroid stimulating hormone (TSI-Ifl); ovine prolactin (PRL) or rat growth hormone (GH). The specificity of these antisera has been previously demonstrated2,3,12. A cell nucleus was considered to be radiolabelled if the number of silver grains was more than 3-fold higher than that present over an equivalent area of extracellular space or cytoplasm. The percentage of cells labelled in the partes distalis, intermedia and nervosa was determined after counting a minimum of 250 cells in each lobe. Similar calculations, based on cell counts of 125 cells, were made on each of the immunostained sections in an effort to assess the percentage of L H , TSH, P R L and G H cells that were targets for D H T .

and retention was observed in 3.2% of the cells of the pars intermedia and 9.6% of the pituicytes in the pars nervosa (Fig. 1). In the pars distalis, 3% of the total cell population retained the [3H]DHT. These cells were all basophilic with no silver grains found in association with the acidophils or with the chromophobes. Combined immunostaining and autoradiographic localization revealed that the primary target cells were those that contained either L H (Fig. 2) or TSH; however, within these two cell populations, only 51% and 10% respectively were radlolabelled. Less than 1% of the somatotrophs and m a m m o trophs displayed silver grains over their nuclei. Specific autoradiographic localization of the [3H]DHT was prevented in the pituitary cells of the guinea pig injected with both the radiolabelled and the unlabelled D H T .

RESULTS

DISCUSSION

Radiolabelling was present over nuclei of cells in all 3 regions of the pituitary gland examined. Uptake

Autoradiographic localization of the ovarian steroid hormones has been reported using the guinea

Fig 1 Autoradiogram of the pars nervosa of a male guinea pig injected with [3H]DHT Two pltmcyte nuclei (arrows) are radlolabelled while several others m the field are unlabelled m&catmg that they did not take up the trmated DHT x 1735 Fig 2. Portion of the pars dlstahs of a male guinea pig rejected with [3H]DHT that was lmmunocytochemlcally stained with antlbo&es to ovme LI-Ifl Two of the 3 lmmunostamed LH cells also bound the radlolabelled androgen (arrows) × 1735

320 pig hypophysislS,21,22. Estrogen was confined to the partes distalis and intermedia18, 21, while the progesterone data appeared equivocal with no specific cellular uptake of the steroid 22. In our study, which represents the first combined autoradiographic/immunocytochemical analysis of the guinea pig hypophysis, the [3H]DHT was present in all 3 lobes of the gland. It is not known whether the cells which retamed the androgen would have also taken up [3H]estradiol. One might presume this to be the case at least with respect to certain cell populations in the pars distalis. For example, in the baboon, we observed that 78% of the LH-secreting cells bound [3H]DHT and 89% retained [3H]estradiol4. It seems from these data that a high percentage of the L H cells probably have receptors for both steroid hormones. As with other species, the main cell type in the guinea pig pars distalis which retained the [3H]DHT was the L H cell The percentage of radiolabelled gonadotrophs, however, was only 51%, which was similar to what was observed in hamsters 17, but considerably less than that found in baboons 4. The absence of D H T in a subpopulation of L H cells suggests a functional heterogeneity among gonadotrophs. This concept was recently suggested following a study of the baboon hypophysis in animals injected with [3H]progesteroneS. The other cell type to retain [3H]DHT in the guinea pig was the thyrotroph. Rat TSH cells as well as those in monkeys and baboons also bind D H T 4,6,15. The interact20ns between D H T and TSH secretion have only recently been shown. In rats that are either intact or castrated, testosterone and dehydroepiandrosterone administration causes an increase in serum TSHa, 20. In contrast, no relationship exists with androgens and TSH in humans 8. In the guinea p~g, we observed uptake and retention of the [3H]DHT by only 3% of the parenchymal cells of the pars distalis. This is a much smaller andro-

REFERENCES 1 Chrlstlanson, D , Rotl, E , Vagenaklc, A G. and Braverman, L E , The sex-related difference in serum thyrotropm concentration Is androgen mediated, Endocrinology, 108 (1981) 529-535 2 Herbert, D C, Identification of the LH and TSH-secretlng cells in the pituitary gland of the rhesus monkey, Cell Tissue Res, 190 (1978) 151-161

gen-sensmve cell population than what is present in other species. In the rat and hamster, for example, 10-15% bind either [3H]testosterone14 or [3H]DHTI7. Similar results are found in mice 16 and baboons 4. The reasons for such differences are unclear. Since the guinea pigs were castrated prior to the onset of the study, there should have been very little endogenous hormone bound to receptor sites to compete with the exogenously administered D H T . Radiolabelling was also found m the guinea pig partes intermedia and nervosa. These data agree well with what has been reported in ratslS, 17, hamsters ~7, monkeys 6 and baboons 4. In mice, D H T is taken up only by the cells of the partes distalls and nervosa 15. Localization of D H T in the pituicytes has at the present time no known functional significance. It may be due to the relationship pituicytes have with glial cells since androgen banding occurs in the latter cells of the cerebellum (unpublished observations). It is clear that marked differences exist with respect to the number of and classes of hypophyseal cells which bind D H T in any given region of the hypophysis as well as in the presence or absence of target cells in the pars intermedia. While the physiological significance of these discrepancies is unknown, it lS advantageous to contmue examining additional mammalian species and to expand our studies to mclude non-mammals with the anticipation that a clearer understanding of these variances will emerge.

ACKNOWLEDGEMENTS The authors are grateful to Cathy Nichols for her technical assistance and to the National Pituitary Agency for supplying the antiserum to human TSHfl. The work was supported in part by N I H Grant NS12933 and NSF Grant PCM8118487.

3 Herbert, D C and Hayashida, T , Hlstologlc identification and lmmunochemical stu&es of prolactm and growth hormone m the primate pituitary gland, Gen. Comp Endocnnol, 24 (1974) 381-397 4 Herbert, D C and Sheridan, P J , Uptake and retention of sex steroids by the baboon pltuatary gland-evidence of sexual dlmorphlsm with respect to dihydrotestosterone, Btol Reprod, 28 (1983) 377-383 5 Herbert, D C and Sheridan, P J , Identification of the pro-

321 gesterone target cells in the female baboon pituitary gland, Biol Reprod., 30 (1984) 479-483. 6 Herbert, D . C , Weaker, F.J. and Sheridan, P.J., Location of 3H-dihydrotestosterone In the pituitary gland of the rhesus monkey, Cell Tissue Res, 215 (1981) 499-504. 7 Herbert, D C , Weaker, F J and Sheridan, P.J., AutoradIographlc demonstration of estrogen uptake by the armadd1o pituitary gland, Expenentta, 37 (1981) 1035-1036 8 Ingbar, S.H and Woeber, K.A., The thyroid gland. In R.H. Willams (Ed.), Textbook of Endocrinology, W B Saunders, Philadelphia, 1981, pp. 117-247. 9 Ison, E J and Sheridan, P J Autoradlography of diffusible substances - - a practical approach, Amer. J Med Technol., 47 (1981) 38-42 10 Keefer, D A and Dryden, G . L , Nuclear uptake of radioactivity by cells of pituitary, brain, uterus, and vagina of the Asian musk shrew (Suncus murmurs) following [3H]estradlol admlmstratlon, Gen Comp. Endocrlnol., 47 (1982) 125-130 11 Keefer, D A., Stumpf, W E and Petrusz, P , Quantitative autoradlographlc assessment of 3H-estradlol uptake in immunocytochemically characterized pItmtary cells, Cell Tissue Res, 166 (1976) 25-35 12 McGlll, J . R , Herbert, D.C. and Hayashida, T , Immunocytochemlcal identification of rat growth hormone cells utihzing antlsera to nonmammahan growth hormones, Gen Comp Endocrmol, 35 (1978) 342-345. 13 Pfaff, D W., Gerlach, J . L , McEwen, B.S , Ferin, M , Carmel, P and Zlmmerman, E.A., Autoradiographic locahzatlon of hormone-concentrating cells in the brain of the female rhesus monkey, J Comp Neurol., 170 (1976) 279-294 14 Sat, M and Stumpf, W E , Cellular and subcellular locahzatlon of radioactivity in the rat pitmtary after injection of 1,2-3H-testosterone using dry autoradiography, Endocrinology, 92 (1973) 631-635 ,

15 Sar, M. and Stumpf, W.E., Simultaneous localization of steroid and peptIde hormones in rat pituitary by combined thaw-mount autoradlography and lmmunohistochemistry: localization of dihydrotestosterone in gonadotropes, thyrotropes and pituicytes, Cell Tissue Res., 203 (1979) 1-7. 16 Sheridan, P . J , The pars nervosa is a target site for androgen in the mouse, Biol. Reprod., 19 (1978) 657-660. 17 Shendan, P.J. and Herbert, D.C., Nuclear uptake and retention of androgen by the pituitary gland of the hamster and the rat, Cell Tissue Res., 206 (1980) 35-40. 18 Stumpf, W E and Sar, M., Autoradiographic localization of estrogen, androgen, progestln and glucocorticosterold in 'target tissues' and 'nontarget tissues'. In J.R Pasqualini ( E d ) , Receptors and Mechamsm of Action of Sterold Hormones, Part 1, Marcel Dekker, New York, 1976, pp 41-84 19 Stumpf, W.E., Sar, M. and Keefer, D A , Localization of hormones in the pituitary: receptor sites for hormones from hypophysial target cells and the brain In A. Tlxler-Vidal and M.G. Farquhar (Eds), The Anterior Pituitary, Academic Press, New York, 1975, pp. 63-82. 20 Tal, E and Sulman, F . G , Dihydroepiandrosterone-induced thyrotrophin release during heat stress in rats, J Endocrmol, 67 (1975) 99-103. 21 Warembourg, M , Radioautographlc.locahzatlon of estrogen-concentrating cells in the brain and pituitary of the guinea pig, Brain Research, 123 (1977) 357-362 22 Warembourg, M., Radloautographic study of the brain and pituitary after [3H]progesterone injection into estrogen-primed ovarlectomized guinea pigs, Neurosct Lett, 7 (1978) 1-5. 23 Warembourg, M , Uptake of 3H labeled synthetic progestin by rat brain and pituitary. A radioautography study, Neurosct Lett, 9 (1978) 329-332.