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The molecular biology of the FSH receptor
J. Steroid Biochem. Molec. Biol. Vol. 53, No. 1-6, pp. 215-218, 1995 Copyright ~;i 1995 Elsevier Science Ltd 0960-0760(95)00049-6 Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00
Pergamon
The M o l e c u l a r B i o l o g y of the F S H R e c e p t o r M i c h a e l D. Griswold,* Leslie H e c k e r t and Carol Linder Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, U.S.A. T h e a c t i o n s o f f o l l i c l e s t i m u l a t i n g h o r m o n e ( F S H ) m e d i a t e d t h r o u g h its r e c e p t o r a r e n e c e s s a r y for t h e p r o p e r f u n c t i o n i n g o f m a m m a l i a n g o n a d s . T h e F S H r e c e p t o r is l o c a l i z e d o n g r a n u l o s a cells o f t h e o v a r y a n d S e r t o l i cells o f t h e t e s t i s . T h e e x p r e s s i o n o f t h e F S H r e c e p t o r ( F S H R ) in S e r t o l i cells v a r i e s in vivo as a f u n c t i o n o f t h e s t a g e o f t h e c y c l e o f t h e s e m i n i f e r o u s e p i t h e l i u m a n d in c u l t u r e as a r e s u l t o f t h e a d d i t i o n o f e x o g e n o u s h o r m o n e s . T h e g e n e f o r t h e F S H r e c e p t o r is l a r g e a n d h a s b e e n s h o w n to b e r e l a t e d in s t r u c t u r e to t h e g e n e s f o r l u t e i n i z i n g h o r m o n e ( L H ) r e c e p t o r a n d t h y r o i d stimulating hormone (TSH) receptor. The promoter region of the gene for FSHR does not contain a T A T A b o x a n d h a s m u l t i p l e t r a n s c r i p t i o n a l s t a r t sites. L e s s t h a n 280 b p o f t h e p r o m o t e r a r e s u f f i c i e n t in t r a n s i e n t t r a n s f e c t i o n a s s a y s to d i r e c t e x p r e s s i o n o f t h e c h l o r a m p h e n i c o l acetyl t r a n s f e r a s e g e n e ( C A T ) in a n u m b e r o f d i f f e r e n t cell t y p e s i n c l u d i n g n o n - g o n a d a l cells. H o w e v e r , t h e promoter does direct the expression of a marker gene only into testis and ovary of transgenic mice.
J. Steroid Biochem. Molec. Biol., Vol. 53, No. 1-6, pp. 215-218, 1995
INTRODUCTION
FSH RECEPTOR (FSHR)
Follicle stimulating h o r m o n e (follitropin; F S H ) is a pituitary glycoprotein h o r m o n e that is necessary for normal reproduction in both male and female mammals. Sertoli cells and granulosa cells are the target cells for the action of F S H in the testes and ovaries, respectively. Sertoli cells provide physical and biochemical support for germ cell development [1,2]. Granulosa cells in the ovary play a role in the overall support of the o v u m and the development of the follicle [3]. According to this scenario F S H indirectly influences spermatogenesis and oogenesis by exerting influences on the corresponding somatic cells. T h e granulosa cells of the ovary and the Sertoli cells of the testis share a n u m b e r of similarities which have been noted previously [4]. Both cell types are necessary for the development and survival of the germ cells prior to ovulation or spermatogenesis. I m m a t u r e and mature granulosa and Sertoli cells have some similar biochemical properties including the synthesis of some of the same proteins such as plasminogen activator and inhibin. Both granulosa cells and i m m a t u r e Sertoli cells are capable of synthesizing estrogens from an exogenous source of androgens [4].
Granulosa and Sertoli cells are target cells for the action of F S H and they are the only cell types which are thought to express the F S H receptor. T h e follicle stimulating h o r m o n e receptor ( F S H R ) , like the receptors for luteinizing hormone ( L H ) / C G and thyroid stimulating hormone ( T S H ) , is a m e m b e r of a superfamily of receptors which act via interactions with G-proteins [5-8]. All of the receptors in this superfamily traverse the plasma m e m b r a n e with seven highly conserved ~-helices oriented with an extracellular amino terminus and an intracellular carboxy terminus [9, 10]. T h e glycoprotein h o r m o n e receptors, L H , F S H and T S H represent a small subclass of this superfamily that have the largest amino terminal domains. T h e c D N A for the F S H receptor was shown to encode a 675 amino acid, 75,000 Da protein with a 348 amino acid external domain [8]. T h e extracellular domains of the glycoprotein h o r m o n e subfamily share moderate sequence similarity that includes a repeated series of conserved 25 residue leucine-rich motifs, (for review see [11]).
FSHR GENE We have characterized the F S H R gene and its prom o t e r [12]. T h e F S H R gene is very large, encompassing at least 85 kb of D N A which is divided into 10 exons often separated by very large introns. T h e first 9 exons are relatively small and code for the amino
Proceedings of the I X International Congress on Hormonal Steroids, Dallas, Texas, U.S.A., 24-29 September 1994. *Correspondence to M. D. Griswold. 215
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terminal extracellular domain which contains the leucine repeat regions. T h e region of the molecule which encodes the seven t r a n s m e m b r a n e spanning regions is contained within a single large exon (exon 10). T h e genes coding for F S H R , T S H R and L H R have a n u m b e r of similarities: (1) they are all very large genes with 10 ( T S H R , F S H R ) or 11 ( L H ) exons; (2) the last exon in each gene codes for the entire m e m brane spanning region; and (3) exons 2-9 code for the amino terminal repeats. T h i s structure suggests that these genes arose from a single gene in which an exon coding for the leucine repeats was duplicated several times and subsequently was combined with the conserved exon for the t r a n s m e m b r a n e region. It has been proposed that these receptors ( F S H R , L H R and T S H R ) constitute a closely related subfamily of the G - p r o t e i n coupled receptors which have a relatively recent evolutionary origin [11]. E X P R E S S I O N OF F S H R IN S E R T O L I A N D G R A N U L O S A CELLS
T h e c D N A probe for the F S H receptor has been used to localize the sites of synthesis and to examine the steady state levels of F S H R m R N A . Sertoli cells, ovary and testis were found to contain two major transcripts of 2.6 and 4.5 kb which hybridized to the F S H receptor D N A sequence [13, 14]. Using probes from the genomic sequence we were able to show that the 4.5 kb transcript has an extended 3' terminus probably resulting from a read through of the first poly A signal sequence. Others have reported that ovarian m R N A analyzed on N o r t h e r n blots had two major F S H R transcripts of 7.0 and 2.5 kb [15] or 2.6 and 5 kb [16]. I n situ hybridization has been used to localize the F S H receptor m R N A exclusively in the granulosa cells in the ovary [16]. In the adult male rat, the expression of the F S H R undergoes a cyclic change in steady state levels [13]. Spermatogenesis in the adult rat is organized into a series of 14 stages which constitute the cycle of the seminiferous epithelium and are defined by the germinal cell composition of the tubules [17]. In the normal rat the 14 stages are present simultaneously at different regions along the length of the tubule and the release of spermatozoa (spermiation) which occurs at stage V I I I is asynchronous. T h e r e is good evidence that the functions of the Sertoli cells vary with the different stages of the cycle. We have utilized a scheme of retinol deprivation and repletion which results in the synchronization of the testis to 3 or 4 related stages of the cycle [18, 19]. Spermatogenesis appears to proceed normally but since the entire testis is in roughly the same stages, spermiation occurs only every 12 to 13 days. We have utilized the retinol synchronization method to obtain synchronized testes which represent all parts of the cycle. T h i s method allows the collection of sufficient amounts of stage synchronized testis to enable the
analysis of low abundance cell products [20]. Utilizing the F S H receptor c D N A probe and m R N A isolated from synchronized testes we were able to measure the steady state levels of F S H R m R N A during the different stages of the cycle of the seminiferous epithelium [13]. We found that the relative levels of F S H R m R N A varied in a cyclic m a n n e r with low levels in stages V - I X and 5-fold higher levels in stages X I I I , X I V , and I. T h i s result correlated very well to recent data from other laboratories which used the dissection method to determine other parameters of F S H action. Altogether these studies suggest that the primary action of F S H in the adult male rat is cyclic in nature and may be confined to stages X I I - I V . We examined the possible regulation of steady state levels of F S H R m R N A by hormones and growth factors that play important roles in spermatogenesis. We examined whether or not F S H or phorbol ester could regulate the F S H R m R N A level. Cultured Sertoli cells from 20-day-old rats were treated with different combinations of hormones, phorbol esters and vitamin A. T h e amount of F S H R m R N A in these cells was then assayed by N o r t h e r n blots. T h e data was normalized to the m R N A levels of actin and clusterin which have been shown to be unchanged in cell culture in the presence of different hormones. We found that after 3 days in culture, F S H , insulin, retinol or testosterone added individually did not alter F S H R m R N A levels (relative to total poly A ÷ m R N A ) . However, addition of all 4 reagents resulted in a 1.5-2-fold increase in the steady state F S H R m R N A levels. Studies from another laboratory have shown that F S H added to cultured Sertoli cells down regulated the level of F S H R m R N A within 4 h of addition of hormone to cultured Sertoli cells [21]. Transcriptional run-on experiments by these same investigators showed that F S H did not inhibit the initiation of transcription of the F S H R gene and that by 16h after addition of hormone the F S H R m R N A levels had returned to normal. T h e authors suggest that these results show that the F S H induced receptor down regulation of F S H R m R N A occurred through a post-transcriptional mechanism. A report which showed that phorbol esters desensitize Sertoli cells to F S H p r o m p t e d the examination of the effect of phorbol esters on the steady state levels of F S H R m R N A [22]. T h e addition of phorbol esters to cultured Sertoli cells results in a rapid and dramatic decrease in F S H R m R N A followed by return to steady state levels [14]. T h e mechanism of this inhibitory action is unknown (transcriptional or post-transcriptional) but the results clearly show that phorbol esters can alter the F S H response of Sertoli cells. 5' F L A N K I N G R E G I O N OF F S H R GENE
Understanding the regulation of the F S H R m R N A at the transcriptional level involves an analysis of the
F S H Receptor p r o m o t e r r e g i o n s associated w i t h the gene. W e have c h a r a c t e r i z e d the 5 ' - f l a n k i n g r e g i o n of the rat F S H R gene [12]. P r i m e r e x t e n s i o n a n d S1 n u c l e a s e experi m e n t s revealed the p r e s e n c e of two m a j o r t r a n s c r i p tional start sites at p o s i t i o n s - 8 0 a n d - 9 8 relative to the t r a n s l a t i o n a l start site. T h e p r o m o t e r r e g i o n i m m e d i a t e l y u p s t r e a m f r o m the t r a n s c r i p t i o n a l start site did n o t c o n t a i n c o n v e n t i o n a l T A T A or C C A A T e l e m e n t s . T h e p r o m o t e r region has characteristics w h i c h are similar to p r o m o t e r s of genes c o d i n g for " h o u s e k e e p i n g " f u n c t i o n s [23]. Characteristics of these p r o m o t e r s i n c l u d e m u l t i p l e t r a n s c r i p t i o n initiation sites, a b s e n c e of T A T A a n d C C A A T boxes, a n d high G C c o n t e n t . T h e 5 ' - f l a n k i n g regions of the L H r e c e p t o r gene a n d the T S H r e c e p t o r gene have also b e e n s e q u e n c e d a n d show similar c h a r a c t e r istics. T h e s e q u e n c e s of these p r o m o t e r regions have a h i g h e r overall G C c o n t e n t t h a n does the c o r r e s p o n d i n g region of the F S H r e c e p t o r gene [24-26]. T h i s p r o m o t e r is of c o n s i d e r a b l e i n t e r e s t since it s h o u l d c o n t a i n cell specific e n h a n c e r e l e m e n t s w h i c h m a y direct e x p r e s s i o n of t r a n s genes i n Sertoli cells or g r a n u l o s a cells. I n o r d e r to analyze the p r o m o t e r we c o n s t r u c t e d several f u s i o n genes c o n t a i n i n g f r o m 280 to 5 kb of D N A 5' to the t r a n s l a t i o n a l start site l i n k e d to the r e p o r t e r gene, c h l o r a m p h e n i c o l acetyltransferase. T h e s e gene c o n s t r u c t s were t r a n s f e c t e d into c u l t u r e d Sertoli cells, C O S - 7 cells a n d a m u r i n e cell line d e r i v e d from Sertoli cells d e s i g n a t e d as M S C - 1 . All of these c o n s t r u c t s actively p r o m o t e d t r a n s c r i p t i o n of the r e p o r t e r gene in p r i m a r y c u l t u r e s of Sertoli cells, N I H 3 T 3 cells, M A - 1 0 cells (a L e y dig cell line) a n d M S C - 1 cells b u t n o t in C O S - 7 cells. T h e s e results showed that the D N A e l e m e n t s r e s p o n s i b l e for t r a n s c r i p t i o n reside in the first 280 b p of the 5 ' - f l a n k i n g r e g i o n a n d that p r o m o t e r activity was n o t cell specific in t r a n s i e n t t r a n s f e c t i o n assays. H o w e v e r , w h e n the 5 kb piece of p r o m o t e r was u s e d to drive the t r a n s c r i p t i o n o f / 3 - g a l a c t o s i d a s e in t r a n s genic a n i m a l s , it was f o u n d that the p r o m o t e r directed e x p r e s s i o n of the gene o n l y into testes or ovaries of t r a n s g e n i c mice. T h u s , even t h o u g h the p r o m o t e r is p r o m i s c u o u s in t r a n s i e n t t r a n s f e c t i o n s it works in a very cell specific m o d e in t r a n s g e n i c mice. T h e a p p a r e n t d i s c r e p a n c y i n the results of the t r a n s i e n t t r a n s f e c t i o n assays a n d the results f r o m the t r a n s g e n i c mice have a n u m b e r of possible explan a t i o n s . O n e p o s s i b i l i t y is that in the t r a n s g e n i c mice the t r a n s g e n e is i n a c t i v a t e d b y m e t h y l a t i o n in n o r m a l l y n o n - e x p r e s s i n g tissues. W e have e x a m i n e d the m e t h y l a t i o n state of the e n d o g e n o u s F S H R gene in Sertoli cells a n d in o t h e r n o n - e x p r e s s i n g tissues. W e e x a m i n e d a specific C C G G s e q u e n c e i n the p r o m o t e r a n d f o u n d it to be n o n - m e t h y l a t e d in Sertoli cells of the male a n d to be m e t h y l a t e d i n all o t h e r tissues we e x a m i n e d [27].
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REFERENCES 1. Griswold M. D., Morales C. and Sylvester S. R.: Molecular biology of the Sertoli cell. Oxf. Rev. Reprod. Biol. 10 (1988) 124-161. 2. Griswold M. D.: Protein secretions of Sertoli cells. Int. Rev. Cytol. 110 (1988) 133-156. 3. Amsterdam A. and Rotmensch S.: Structure-function relationships during granulosa cell differentiation. Endocrine Rev. 8 (1987) 309-337. 4. Fritz I. B.: Comparison of granulosa and Sertoli cells at various stages of maturation: similarities and differences. Adv. Exp. Med. Biol. 147 (1982) 357-384. 5. Parmentiar M.: Molecular cloning of the thyrotropin receptor. Science 246 (1989) 1620-1622. 6. Loosfelt H., Misrahi M., Atger M., Salesse R., Thi M. T. V. H L., Jolivet A., Guiochon-Mantel A., Sar S., Jallal B., Garnier J. and Milgrom E.: Cloning and sequencing of porcine LH-hCG receptor eDNA: variants lacking transmembrane domain. Science 245 (1989) 525-528. 7. McFarland K. C., Sprengel R., Phillips H. S., K0hler M., Rosemblit N., Nikolics K., Segaloff D. L. and Seeburg P. H.: Lutropin-choriogonadotropin receptor: an unusual member of the G-protein-coupled receptor family. Science 245 (1989) 494-499. 8. Sprengel R., Braun T., Nikolics K., Segaloff D, L. and Seeburg P. H.: The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Molec. Endocr. 4, (1990) 525-530. 9. Johnson G. L. and Dhanasedaran N.: The G protein family and their interaction with receptors. Endocrine Rev. 10 (1989) 317-331. 10. O'Dowd B. F., Lefkowitz R. J. and Caron M. G.: Structure of the adrenergic and related receptors. Ann. Rev. Neurosci. 12 (1989) 67-83. 11. Vassart G., Parmentier M., Libert F. and Dumont J.: Molecular genetics of the thyrotropin receptor. Trends Endocr. Metab. 2 (1991) 151-156. 12. Heckert L. L., Daley I. and Griswold M. D.: Structural organization of the follicle hormone receptor gene. Molec. Endocr. 6 (1992) 70-80. 13. Heckert L. L. and Griswold M. D. Expression of folliclestimulating hormone receptor mRNA in rat testes and Sertoli cells. Molec. Endocr. 5 (1991) 670-677. 14. Heckert L. L. and Griswold M. D.: Expression of the FSH receptor in the testis. Rec. Prog. Horm. Res. 48 (1992) 61-78. 15. LaPolt P. S., Tilly J. L., Aihara T., Nishimori K. and Hsueh A.: Gonadotropin-induced up and down regulation of ovarian follicle stimulating hormone (FSH) receptor gene expression in immature rats: effects of pregnant mare's serum gonadotropin, human chorionic gonadotropin and recombinant FSH. Endocrinology 130 (1992) 1289-1295. 16. Camp T. A., Rahal J. O. and Mayo K. E.: Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Molec. Endocr. 5 (1991) 1405-1417. 17. Leblond C. P. and Clermont Y.: Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N . Y . Acad. Sci. 55 (1952) 548-573. 18. Morales C. R. and Griswold M. D.: Retinol induces stage synchronization in seminiferous tubules of vitamin A deficient rats. Ann. N . Y . Acad. Sci. 513 (1987) 292-293. 19. Morales C., Hugly S. and Griswold M. D.: Stage-dependent levels of specific mRNA transcripts in Sertoli cells. Biol. Reprod. 36 (1987) 1035-1046. 20. Morales C. R., Alcivar A. A., Hecht N. B. and Griswold M. D.: Specific mRNAs in Sertoli and germinal cells of testes from stage synchronized rats. Molec. Endocr. 3 (1989) 725-733. 21. Themmen A., Blok L., Post M., Baarends W., Hoogerbrugge J., Parmentier M., Vassart G. and Grootegood A.: Follitropin receptor down regulation involves a cAMP-dependent posttranscriptional decrease of receptor mRNA expression. Molec. Cell. Endocr. 78 (1991) R7-R13. 22. Monaco L. and Conti M.: Inhibition by phorbol esters and other tumor promoters of the response of the Sertoli cell to FSH: evidence for dual site of action. Molec. Cell Endocr. 49 (1987) 227-236.
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23. Dynan W. S.: Promoters for housekeeping genes. Trends Genet. 2 (1986) 196-197. 24. Tsai-Morris C. H., Buczko E., Wei W., Xie X. Z. and Dufan M. L.: Structural organization of the rat leuteinizing hormone (LH) receptor gene. J. Biol. Chem. 266 (1991) 11,355-11,358. 25. Koo ¥. B., Ji I., Slaughter R. G. and Ji T. H.: Structure of the leuteinizing hormone receptor: gene and multiple
exons of the coding sequence. Endocrinology 128 (1991) 2297-2650. 26. Ikuyama S., Niller H., Shimura H., Akamizu T. and Kohn L. D.: Characterization of the 5~-flanking region of the rat thyrotropin receptor gene. Molec. Endocr. 6 (1992) 793-804. 27. Linder C. C., Heckert L. L., Goetz T. L. and Griswold M. D.: Follicle stimulating hormone receptor gene promoter activity. Endocrine 2 (1994) 957-966.
Pergamon
The M o l e c u l a r B i o l o g y of the F S H R e c e p t o r M i c h a e l D. Griswold,* Leslie H e c k e r t and Carol Linder Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, U.S.A. T h e a c t i o n s o f f o l l i c l e s t i m u l a t i n g h o r m o n e ( F S H ) m e d i a t e d t h r o u g h its r e c e p t o r a r e n e c e s s a r y for t h e p r o p e r f u n c t i o n i n g o f m a m m a l i a n g o n a d s . T h e F S H r e c e p t o r is l o c a l i z e d o n g r a n u l o s a cells o f t h e o v a r y a n d S e r t o l i cells o f t h e t e s t i s . T h e e x p r e s s i o n o f t h e F S H r e c e p t o r ( F S H R ) in S e r t o l i cells v a r i e s in vivo as a f u n c t i o n o f t h e s t a g e o f t h e c y c l e o f t h e s e m i n i f e r o u s e p i t h e l i u m a n d in c u l t u r e as a r e s u l t o f t h e a d d i t i o n o f e x o g e n o u s h o r m o n e s . T h e g e n e f o r t h e F S H r e c e p t o r is l a r g e a n d h a s b e e n s h o w n to b e r e l a t e d in s t r u c t u r e to t h e g e n e s f o r l u t e i n i z i n g h o r m o n e ( L H ) r e c e p t o r a n d t h y r o i d stimulating hormone (TSH) receptor. The promoter region of the gene for FSHR does not contain a T A T A b o x a n d h a s m u l t i p l e t r a n s c r i p t i o n a l s t a r t sites. L e s s t h a n 280 b p o f t h e p r o m o t e r a r e s u f f i c i e n t in t r a n s i e n t t r a n s f e c t i o n a s s a y s to d i r e c t e x p r e s s i o n o f t h e c h l o r a m p h e n i c o l acetyl t r a n s f e r a s e g e n e ( C A T ) in a n u m b e r o f d i f f e r e n t cell t y p e s i n c l u d i n g n o n - g o n a d a l cells. H o w e v e r , t h e promoter does direct the expression of a marker gene only into testis and ovary of transgenic mice.
J. Steroid Biochem. Molec. Biol., Vol. 53, No. 1-6, pp. 215-218, 1995
INTRODUCTION
FSH RECEPTOR (FSHR)
Follicle stimulating h o r m o n e (follitropin; F S H ) is a pituitary glycoprotein h o r m o n e that is necessary for normal reproduction in both male and female mammals. Sertoli cells and granulosa cells are the target cells for the action of F S H in the testes and ovaries, respectively. Sertoli cells provide physical and biochemical support for germ cell development [1,2]. Granulosa cells in the ovary play a role in the overall support of the o v u m and the development of the follicle [3]. According to this scenario F S H indirectly influences spermatogenesis and oogenesis by exerting influences on the corresponding somatic cells. T h e granulosa cells of the ovary and the Sertoli cells of the testis share a n u m b e r of similarities which have been noted previously [4]. Both cell types are necessary for the development and survival of the germ cells prior to ovulation or spermatogenesis. I m m a t u r e and mature granulosa and Sertoli cells have some similar biochemical properties including the synthesis of some of the same proteins such as plasminogen activator and inhibin. Both granulosa cells and i m m a t u r e Sertoli cells are capable of synthesizing estrogens from an exogenous source of androgens [4].
Granulosa and Sertoli cells are target cells for the action of F S H and they are the only cell types which are thought to express the F S H receptor. T h e follicle stimulating h o r m o n e receptor ( F S H R ) , like the receptors for luteinizing hormone ( L H ) / C G and thyroid stimulating hormone ( T S H ) , is a m e m b e r of a superfamily of receptors which act via interactions with G-proteins [5-8]. All of the receptors in this superfamily traverse the plasma m e m b r a n e with seven highly conserved ~-helices oriented with an extracellular amino terminus and an intracellular carboxy terminus [9, 10]. T h e glycoprotein h o r m o n e receptors, L H , F S H and T S H represent a small subclass of this superfamily that have the largest amino terminal domains. T h e c D N A for the F S H receptor was shown to encode a 675 amino acid, 75,000 Da protein with a 348 amino acid external domain [8]. T h e extracellular domains of the glycoprotein h o r m o n e subfamily share moderate sequence similarity that includes a repeated series of conserved 25 residue leucine-rich motifs, (for review see [11]).
FSHR GENE We have characterized the F S H R gene and its prom o t e r [12]. T h e F S H R gene is very large, encompassing at least 85 kb of D N A which is divided into 10 exons often separated by very large introns. T h e first 9 exons are relatively small and code for the amino
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terminal extracellular domain which contains the leucine repeat regions. T h e region of the molecule which encodes the seven t r a n s m e m b r a n e spanning regions is contained within a single large exon (exon 10). T h e genes coding for F S H R , T S H R and L H R have a n u m b e r of similarities: (1) they are all very large genes with 10 ( T S H R , F S H R ) or 11 ( L H ) exons; (2) the last exon in each gene codes for the entire m e m brane spanning region; and (3) exons 2-9 code for the amino terminal repeats. T h i s structure suggests that these genes arose from a single gene in which an exon coding for the leucine repeats was duplicated several times and subsequently was combined with the conserved exon for the t r a n s m e m b r a n e region. It has been proposed that these receptors ( F S H R , L H R and T S H R ) constitute a closely related subfamily of the G - p r o t e i n coupled receptors which have a relatively recent evolutionary origin [11]. E X P R E S S I O N OF F S H R IN S E R T O L I A N D G R A N U L O S A CELLS
T h e c D N A probe for the F S H receptor has been used to localize the sites of synthesis and to examine the steady state levels of F S H R m R N A . Sertoli cells, ovary and testis were found to contain two major transcripts of 2.6 and 4.5 kb which hybridized to the F S H receptor D N A sequence [13, 14]. Using probes from the genomic sequence we were able to show that the 4.5 kb transcript has an extended 3' terminus probably resulting from a read through of the first poly A signal sequence. Others have reported that ovarian m R N A analyzed on N o r t h e r n blots had two major F S H R transcripts of 7.0 and 2.5 kb [15] or 2.6 and 5 kb [16]. I n situ hybridization has been used to localize the F S H receptor m R N A exclusively in the granulosa cells in the ovary [16]. In the adult male rat, the expression of the F S H R undergoes a cyclic change in steady state levels [13]. Spermatogenesis in the adult rat is organized into a series of 14 stages which constitute the cycle of the seminiferous epithelium and are defined by the germinal cell composition of the tubules [17]. In the normal rat the 14 stages are present simultaneously at different regions along the length of the tubule and the release of spermatozoa (spermiation) which occurs at stage V I I I is asynchronous. T h e r e is good evidence that the functions of the Sertoli cells vary with the different stages of the cycle. We have utilized a scheme of retinol deprivation and repletion which results in the synchronization of the testis to 3 or 4 related stages of the cycle [18, 19]. Spermatogenesis appears to proceed normally but since the entire testis is in roughly the same stages, spermiation occurs only every 12 to 13 days. We have utilized the retinol synchronization method to obtain synchronized testes which represent all parts of the cycle. T h i s method allows the collection of sufficient amounts of stage synchronized testis to enable the
analysis of low abundance cell products [20]. Utilizing the F S H receptor c D N A probe and m R N A isolated from synchronized testes we were able to measure the steady state levels of F S H R m R N A during the different stages of the cycle of the seminiferous epithelium [13]. We found that the relative levels of F S H R m R N A varied in a cyclic m a n n e r with low levels in stages V - I X and 5-fold higher levels in stages X I I I , X I V , and I. T h i s result correlated very well to recent data from other laboratories which used the dissection method to determine other parameters of F S H action. Altogether these studies suggest that the primary action of F S H in the adult male rat is cyclic in nature and may be confined to stages X I I - I V . We examined the possible regulation of steady state levels of F S H R m R N A by hormones and growth factors that play important roles in spermatogenesis. We examined whether or not F S H or phorbol ester could regulate the F S H R m R N A level. Cultured Sertoli cells from 20-day-old rats were treated with different combinations of hormones, phorbol esters and vitamin A. T h e amount of F S H R m R N A in these cells was then assayed by N o r t h e r n blots. T h e data was normalized to the m R N A levels of actin and clusterin which have been shown to be unchanged in cell culture in the presence of different hormones. We found that after 3 days in culture, F S H , insulin, retinol or testosterone added individually did not alter F S H R m R N A levels (relative to total poly A ÷ m R N A ) . However, addition of all 4 reagents resulted in a 1.5-2-fold increase in the steady state F S H R m R N A levels. Studies from another laboratory have shown that F S H added to cultured Sertoli cells down regulated the level of F S H R m R N A within 4 h of addition of hormone to cultured Sertoli cells [21]. Transcriptional run-on experiments by these same investigators showed that F S H did not inhibit the initiation of transcription of the F S H R gene and that by 16h after addition of hormone the F S H R m R N A levels had returned to normal. T h e authors suggest that these results show that the F S H induced receptor down regulation of F S H R m R N A occurred through a post-transcriptional mechanism. A report which showed that phorbol esters desensitize Sertoli cells to F S H p r o m p t e d the examination of the effect of phorbol esters on the steady state levels of F S H R m R N A [22]. T h e addition of phorbol esters to cultured Sertoli cells results in a rapid and dramatic decrease in F S H R m R N A followed by return to steady state levels [14]. T h e mechanism of this inhibitory action is unknown (transcriptional or post-transcriptional) but the results clearly show that phorbol esters can alter the F S H response of Sertoli cells. 5' F L A N K I N G R E G I O N OF F S H R GENE
Understanding the regulation of the F S H R m R N A at the transcriptional level involves an analysis of the
F S H Receptor p r o m o t e r r e g i o n s associated w i t h the gene. W e have c h a r a c t e r i z e d the 5 ' - f l a n k i n g r e g i o n of the rat F S H R gene [12]. P r i m e r e x t e n s i o n a n d S1 n u c l e a s e experi m e n t s revealed the p r e s e n c e of two m a j o r t r a n s c r i p tional start sites at p o s i t i o n s - 8 0 a n d - 9 8 relative to the t r a n s l a t i o n a l start site. T h e p r o m o t e r r e g i o n i m m e d i a t e l y u p s t r e a m f r o m the t r a n s c r i p t i o n a l start site did n o t c o n t a i n c o n v e n t i o n a l T A T A or C C A A T e l e m e n t s . T h e p r o m o t e r region has characteristics w h i c h are similar to p r o m o t e r s of genes c o d i n g for " h o u s e k e e p i n g " f u n c t i o n s [23]. Characteristics of these p r o m o t e r s i n c l u d e m u l t i p l e t r a n s c r i p t i o n initiation sites, a b s e n c e of T A T A a n d C C A A T boxes, a n d high G C c o n t e n t . T h e 5 ' - f l a n k i n g regions of the L H r e c e p t o r gene a n d the T S H r e c e p t o r gene have also b e e n s e q u e n c e d a n d show similar c h a r a c t e r istics. T h e s e q u e n c e s of these p r o m o t e r regions have a h i g h e r overall G C c o n t e n t t h a n does the c o r r e s p o n d i n g region of the F S H r e c e p t o r gene [24-26]. T h i s p r o m o t e r is of c o n s i d e r a b l e i n t e r e s t since it s h o u l d c o n t a i n cell specific e n h a n c e r e l e m e n t s w h i c h m a y direct e x p r e s s i o n of t r a n s genes i n Sertoli cells or g r a n u l o s a cells. I n o r d e r to analyze the p r o m o t e r we c o n s t r u c t e d several f u s i o n genes c o n t a i n i n g f r o m 280 to 5 kb of D N A 5' to the t r a n s l a t i o n a l start site l i n k e d to the r e p o r t e r gene, c h l o r a m p h e n i c o l acetyltransferase. T h e s e gene c o n s t r u c t s were t r a n s f e c t e d into c u l t u r e d Sertoli cells, C O S - 7 cells a n d a m u r i n e cell line d e r i v e d from Sertoli cells d e s i g n a t e d as M S C - 1 . All of these c o n s t r u c t s actively p r o m o t e d t r a n s c r i p t i o n of the r e p o r t e r gene in p r i m a r y c u l t u r e s of Sertoli cells, N I H 3 T 3 cells, M A - 1 0 cells (a L e y dig cell line) a n d M S C - 1 cells b u t n o t in C O S - 7 cells. T h e s e results showed that the D N A e l e m e n t s r e s p o n s i b l e for t r a n s c r i p t i o n reside in the first 280 b p of the 5 ' - f l a n k i n g r e g i o n a n d that p r o m o t e r activity was n o t cell specific in t r a n s i e n t t r a n s f e c t i o n assays. H o w e v e r , w h e n the 5 kb piece of p r o m o t e r was u s e d to drive the t r a n s c r i p t i o n o f / 3 - g a l a c t o s i d a s e in t r a n s genic a n i m a l s , it was f o u n d that the p r o m o t e r directed e x p r e s s i o n of the gene o n l y into testes or ovaries of t r a n s g e n i c mice. T h u s , even t h o u g h the p r o m o t e r is p r o m i s c u o u s in t r a n s i e n t t r a n s f e c t i o n s it works in a very cell specific m o d e in t r a n s g e n i c mice. T h e a p p a r e n t d i s c r e p a n c y i n the results of the t r a n s i e n t t r a n s f e c t i o n assays a n d the results f r o m the t r a n s g e n i c mice have a n u m b e r of possible explan a t i o n s . O n e p o s s i b i l i t y is that in the t r a n s g e n i c mice the t r a n s g e n e is i n a c t i v a t e d b y m e t h y l a t i o n in n o r m a l l y n o n - e x p r e s s i n g tissues. W e have e x a m i n e d the m e t h y l a t i o n state of the e n d o g e n o u s F S H R gene in Sertoli cells a n d in o t h e r n o n - e x p r e s s i n g tissues. W e e x a m i n e d a specific C C G G s e q u e n c e i n the p r o m o t e r a n d f o u n d it to be n o n - m e t h y l a t e d in Sertoli cells of the male a n d to be m e t h y l a t e d i n all o t h e r tissues we e x a m i n e d [27].
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