chorionic gonadotropin receptor gene in rat prostates

P*ad E ellular

ndocrinology

ELSEVIER

Molecular and Cellular Endocrinology I I 1 (1995) R9-R 12

Rapid paper

Novel expression of luteinizing hormonekhorionic gene in rat prostates

gonadotropin receptor

Y.X. Tao, Z.M. Lei, S.H. Woodworth’, Ch.V. Rao* Depurfment

qf Obstetrics

and Gynecology,

University

ofLouisville

School

#Medicine,

Louisville,

KY 40292,

USA

Received 13 March 1995; accepted 13 April 1995

Abstract The reverse transcription-polymerase chain reaction (RT-PCR) amplified an expected 255 bp luteinizing hormone/chorionic gonadotropin (LHKG) receptor sequence from rat prostates. Northern blotting demonstrated that prostates contain 4.3, 3.3, 2.6, I .8, 0.8 and 0.2 kb LHKG receptor transcripts. Western immunoblotting and ligand blotting demonstrated that prostates also contain an 80 kDa receptor protein which can bind 1251-labeled hCG and this binding was inhibited by excess unlabeled hCG. In situ hybridization and immunocytochemistry revealed that while the receptors are most abundant in epithelial cells, they are scarcely found in the stroma. The ventral lobe contained more receptors than the lateral lobe and the receptors in peripheral acini of the ventral lobe are higher which progressively decreased towards central acini. In summary, prostate glands express LHKG receptor gene. The cellular, topographical and lobular distribution of receptors suggest that LH may directly regulate prostate functions. Keywords:

Luteinizing

hormone/chorionic

gonadotropin

receptor sequence; Prostate; Rat

1. Introduction Luteinizing

hormone/chorionic

gonadotropin

(LH/CG)

receptors which were once thought to be present only in gonads, have now also been found in several non-gonadal female reproductive tissues including uterus (Reshef et al., 1990; Lei et al., 1993a,b; Rao et al., 1993). Since the prostatic utricle has a developmental similarity to the uterus, we felt that non-gonadal reproductive tissues of

male may also express LWCG receptor gene. For this reason, as well as several others, we used adult rat prostates to investigate the above possibility. First, growth and

regression of prostate has been shown to be associated with changes in LH levels, i.e. castration and androgen replacement (Cunha et al., 1987). These growth changes in prostate were thought to be due to changes in androgen

levels. If the LHKG

receptors are present in prostate,

* Corresponding author. ’ Current address: Medcenter One, 300 North 7th Street, Bismarck, ND 58502, USA. 0303-7207/95/$09.50 0 1995 Elsevier SSDl 0303-7207(95)03564-N

Science

Ireland

Ltd. All rights

reserved

then it is possible that these growth changes could at least partly be due to changes in LH levels. Second, benign prostatic hypertrophy and prostate cancers occur in men during the years when the total and free androgen levels are decreasing (Davidson et al., 1983; Tenover et al., 1987) and LH levels are increasing. Third, even though androgens are generally believed to be essential, they alone are not known to be sufficient for growth, development and function of the prostate glands (Cunha et al., 1987; Rui and Purvis, 1988; Davies and Eaton, 1991; Luke and Coffey, 1994). Finally, growth factors are known to be involved in the regulation of prostate functions (Davies and Eaton, 1991; Luke and Coffey, 1994). Recently, CC has been shown to belong to a family of cystine-knot growth factors (Lapthorn et al., 1994). This makes hCG and LH, another member of the same family, as potential regulators of prostate functions if it contains LWCG receptors. In view of the above reasons, a demonstration that prostates express LHKG receptor gene could have tremendous relevance to both physiologic and pathologic regulation of prostate functions.

RlO

2. Materials

Y.X. Tao et al. I Molecular

and Cellular

and methods

Adult rat prostate glands were purchased from Harlan Bioproducts for Science (Indianapolis, IN). The sources of highly purified hCG (CR- 127; 14 900 IU/mg), LWCG receptor cDNA, polyclonal LWCG receptor antibody and the receptor peptide (amino acids 15-38) are the same as previously described (Lei et al., 1993a,b; Rao et al.,

Endocrinology

I1 I (1995)

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Immunocytochemistry was performed using 5 pm thick sections and 1:300 dilution of receptor antibody (Reshef et al., 1990). For the procedural controls, receptor antibody was omitted or pre-immune rabbit serum was used in place of receptor antibody. One section from each prostate gland was stained with hematoxylin and eosin to differentiate ventral from lateral lobes using criteria of Jesik et al. (1982).

1993).

The RT-PCR was performed with modifications from a previously described nested procedure (Lei et al., 1993a). Briefly, 5 ,ug of total RNA isolated from prostates was reverse transcribed into cDNA. One-fifth of the reaction mixture was amplified in 35 cycles using primers corresponding to rat receptor cDNA sequence of 846 + 866 bp at the .5’-end (primer 1) and 1101 + 1081 bp at the 3’-end (primer 2). The sequences of the primers are: Primer 1 = 5’-AATICACGAGCCTCCTGGTCPrimer 2 = 5’-GCATCTGGTTCTGGAGCACA-3’

3’

The identity of the amplified product was established by Southern blotting using a full length LWCG receptor cDNA. The controls consisted of omission of template or omission of reverse transcriptase. The size of the amplified product was determined from comparison with a 123 bp DNA ladder run in an adjacent lane. Northern blotting was performed as previously described using 5-1Opg mRNA isolated from prostates, 32P-labeled riboprobe transcribed from LWCG receptor cDNA with an in vitro transcription kit from Promega (Madison, WI) and high stringency washing conditions (Sambrook et al., 1989; Lei et al., 1993a). The washed blots were exposed to Kodak XAR-2 film with intensifying screens in the dark at -80°C for l-2 weeks. Western immunoblotting was performed as previously described using 5Opg of homogenate protein (Dunn, 1986; Lei et al., 1993a). The receptor antibody was used at a dilution of 1: 1000. For a procedural control, receptor antibody pre-absorbed with excess receptor peptide was used. Ligand blotting was performed using 100,ug protein of 16 000 X g pellet of homogenates (Keinanen et al., 1987; Lei et al., 1993a). Unlabeled hCG was radioiodinated by a lactoperoxidase technique to a specific activity of 88.3,&i/pg (Rao et al., 1977). The blots were incubated overnight at 4°C with 1 x lo6 cpm/ml of lz51-labeled hCG in the presence and absence of 25,uglml unlabeled hCG. Then the blots were extensively washed and exposed to Kodak XAR-2 film with intensifying screens in the dark at -80°C for 1-2 weeks. In situ hybridization was performed as previously described using 35S-labeled riboprobes transcribed from LWCG receptor cDNA (Angerer et al., 1987; Lei et al., 1993a). Hybridization with 35S-labeled sense riboprobe served as a procedural control. The washed slides were exposed to the emulsion in the dark at 4°C for 7-10 days.

3. Results

We first investigated the presence of LWCG receptor transcripts in prostates by RT-PCR/Southern blotting. The results showed that RT-PCR amplified an expected 255 bp LWCG receptor sequence from prostates (Fig. IA, lanes 1 and 2). As expected, this procedure amplified a receptor sequence from rat ovary used as a positive control tissue (Fig. lA, lane 4), but not in a procedural control in which either the template (lane 3) or reverse transcriptase was omitted. The Southern blotting confirmed that the amplified sequence is indeed that of LWCG receptors. Northern blotting showed that prostates contain 4.3, 3.3, 2.6, 1.8, 0.8 and 0.2 kb receptor transcripts (Fig. lB, lane 3). As expected, rat ovaries (Fig. lB, lane 1) and testes (lane 2) used as positive control tissues, contained a

A 255 bp

l

D

C

80 KDa*

30 KDa12

Fig. I. RT-PCREouthem blotting’(A), northern blotting (B), western immunoblotting (C) and ligand blotting (D) for LHKG receptors in rat prostates. The identity of lanes in each panel is as follows. In (A), lanes I and 2, prostates; lane 3, RNA omission control; lane 4, rat ovary. In (B), lane 1, rat ovary; lane 2. rat testes; lane 3, prostate; lane 4, rat liver. In (C), lane 1, unabsorbed receptor antibody; lane 2, receptor antibody pre-absorbed with receptor peptide. In (D), lane 1, ‘%labeled hCG alone; lane 2, I*%-hCG + unlabeled hCG.

Y.X. Tao et al. I Moleculur

and Cellulur

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I11 (1995)

R9-RI2

Fig. 2. In situ hybridization (a-c) and immunocytochemistry (d-g) for LHKG receptors in rat prostates. (b) An in situ hyhridization control. The Urows in (c) and (d) indicate a decrease of hybridization signals or receptor immunostaining from peripheral to central acini of ventral lobe. Arrowheads in (e) and (g) show acinar epithelial cells of the lateral lobe. (f) and (g) are immunocytochemistry controls for ventral and lateral lobes, respectively. Magnification: (a,b) x600; (c-g) x 25.

higher abundance of multiple receptor transcripts and rat liver (lane 4) did not contain any. Using equal amounts of poly(A)+ RNA and the same exposure times to X-ray film, we found that positive control tissues contain a much higher abundance of transcripts than prostates. Even though hybridization bands are not discrete due to overexposure, it is apparent that gonadal tissues contain some transcripts that are not present in prostates and vice versa. Western immunoblotting showed that prostates contained 80 kDa and 30 kDa proteins (Fig. lC, lane 1). Preabsorption of the receptor antibody with the excess receptor peptide resulted in the disappearance of an 80 kDa but not the 30 kDa protein (lane 2). Ligand blotting demonstrated that an 80 kDa but not 30 kDa protein can bind i251-labeled hCG (Fig. lD, lane 1) and the binding was inhibited by excess unlabeled hCG (lane 2). In situ hybridization showed that hybridization signals, indicating the presence of receptor transcripts, are present in epithelial cells (Fig. 2a). These signals are dramatically reduced in the sense control (Fig. 2b). The hybridization signals due to antisense probe are higher in peripheral acini and decreased towards central acini (Fig. 2~).

Immunocytochemistry showed that LWCG receptor immunostaining is most abundant in epithelial cells (Fig. 2d). The stroma, on the other hand, contained very little or no receptor immunostaining. The receptor immunostaining is the highest in peripheral acini and progressively decreased towards the central acini of the ventral lobe (Fig. 2d). The receptor immunostaining is higher in ventral compared to the lateral lobe (Fig. 2e versus d). The receptor immunostaining in the epithelial cells of ventral (Fig. 2f) and lateral (Fig. 2g) lobes greatly decreased in the procedural controls. Despite this decrease, the staining in acinar contents of lateral lobe remained about the same, suggesting that it is non-specific (Fig. 2%). 4. Discussion Both RT-PCRKouthern blotting and Northern analysis demonstrated that rat prostates contain LH/CG receptor transcripts. However, the relative abundance in prostates was lower than in gonadal tissues. Prostates also contain an 80 kDa protein which can bind lz51-labeled hCG. We recently found that normal human prostates, benign prostatic hypertrophy and prostate cancer tissues

RI2

Y.X. Tuo et al. I Molecular

and Cellular

also contain LWCG receptor mRNA transcripts and receptor protein (Tao et al., unpublished data). The presence of LWCG receptors suggests that LH may directly regulate prostate functions. Although we do not know what functions LH can regulate, the following findings suggest that LWCG may regulate some of them in prostates. For example, epithelial cells in prostate, which are primary sites of LWCG receptor expression, contain high levels of androgen receptors (Sar et al., 1970; Bruner-Losand et al., 1984; Tan et al., 1988; Liao et al., 1989), secrete prostatic acid phosphatase (Ronnberg et al., 1981) and prostate specific antigen (Papsidero et al., 1981). These cells show marked regressive changes following castration and regenerate rapidly after androgen replacement (Huttunen et al., 1981; English et al., 1985). Peripheral acini, which showed higher LHKG receptor expression than central acini, undergo marked changes following androgen deprivation and subsequent androgen replacement (Cunha et al., 1987). Ventral lobes, which showed higher LHKG receptor expression, are more sensitive than the lateral lobes to the above hormonal manipulations (Lee, 1981). Since we now know that prostates contain LWCG receptors, it is possible that increased LH levels may play a role in changes seen in prostate that are traditionally attributed solely to changes in androgens. In summary, rat prostates express LWCG receptors. The cellular, topographical and lobular distribution of receptors along with the previous findings, suggest that LH may directly regulate the functions of prostate. Such a possible regulation may have relevance to physiology and pathology of the prostate gland. References Angerer, L.M.. Cox, K.H. and Angerer, R.C. (1987) Methods Enzymol. 152,64966 I. Bruner-Lorand, J., Mechaber, D., Zwick, A., Hechter, 0.. Eychene, B., Bauleiu, E.E. and Robel, P. (1984) Prostate 5, 231-254. Cunha, G.R., Donjncour. A.A., Cooke, P.S., Mee, S., Bigsby, R.M.. Higgins. S.J. and Sugimura. Y. (1987) Endocr. Rev. 8, 338-362.

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Davidson, J.M., Chen, J.J., Crapo, L., Gray, G.D., Greenleaf, W.J. and Catania. J.A. (1983) J. Clin. Endocrinol. Metab. 57.71-77. Davies, P. and Eaton, CL. (1991) J. Endocrinol. 131.5-17. Dunn. S.D. (1986) Anal. Biochem. 157, 144-153. English, H.F.. Drago, J.R. and Santen, R.J. (1985) Prostate 7, 41-45. Huttunen, E., Romppanen, T. and Helminen, H.J. (1981) J. Anat. 132, 357-370. Jesik, C.J., Holland, J.M. and Lee, C. (1982) Prostate 3. 81-97. Keinanen, K.P., Kellokumpu. S., Metsikko, M.K. and Rajaniemi, H.J. (1987) 1. Biol. Chem. 262,7920-7926. Lapthorn, A.J., Harris, D.C., LittleJohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. and Isaacs, N.W. (1994) Nature 369,455-t6 1. Lee, C. (1981) in The Prostatic Cell: Structure and Function, Part A: Morphologic, Secretory and Biochemical Aspects (Murphy, G.P., Sandberg, A.A. and Karr, J.P., eds.), pp. 145-159, Liss, New York. Lei. Z.M., Rao, Ch.V., Kornyei, J.L., Licht, P. and Hiatt, ES. (1993a) Endocrinology 132, 2262-2270. Lei. Z.M., Toth, P., Rao, Ch.V. and Pridham, D. (1993b) J. Clin. Endocrinol. Metab. 77, 863-872. Liao, S.S., Kokontis, J., Sai, T. and Hiipakka, R.A. (1989) J. Steroid Biochem. 34.41-51. Luke, M.C. and Coffey, D.S. (1994) in The Physiology of Reproduction, 2nd edn. (Knobil, E. and Neill, J.D., eds.), pp. 1435-1487, Raven Press, New York. Papsidero, L.D., Kuriyama, M., Wang, M.C., Horozewicz, J., Leong, S.S., Valenzuela. L.. Murphy, G.B. and Chu, M. (1981) J. Natl. Cancer Inst. 66,3742. Rao, Ch.V., Griffin, L.P. and Carman, Jr., F.R. (1977) Am. J. Obstet. Gynecol. 128, 146-153. Rao, Ch.V., Li, X., Toth, P., Lei. Z.M. and Cook, V.D. (1993) J. Clin. Endocrinol. Metab. 77, 1706-1714. Reshef, E., Lei, Z.M., Rao, Ch.V., Pridham, D.D., Chegini, N. and Luborsky, J.L. (1990) .I. Clin. Endocrinol. Metab. 70.421430. Ronnberg, L., Vihko, P., Sajanti, E. and Vihko, R. (1981) Int. J. Androl. 4, 372-378. Rui, H. and Purvis K. (1988) Stand. J. Urol. Nephrol. Suppl. 107, 3238. Sambrook, J., Fritsch. E.F. and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, 2nd edn., pp. 7.39-7.57, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sar. M., Liao, S. and Stumpf, W.E. (1970) Endocrinology 86, 10081011. Tan, J.-A., Joseph, D.R., Quarmby, V.E.. Lubahn, D.B., Sar, M., French, F.S. and Wilson, E.M. (1988) Mol. Endocrinol. 12, 12761285. Tenover, J.S., Matsumoto, A.M., Plymate, S.R. and Bremner, W.J. (1987) J. Clin. Endocrinol. Metab. 65, 1118-l 126.