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Follicle-stimulating hormone receptor mRNA in the mouse ovary during post-natal development in the normal mouse and in the adult hypogonadal (hpg) mouse: structure of alternate transcripts
Molwular
and
Cellular
Endocrinology
ELSEVIER
Molecular
and Cellular
Endocrinology
101 (1994) 197-201
Follicle-stimulating hormone receptor mRNA in the mouse ovary during post-natal development in the normal mouse and in the adult hypogonadal ( hpg) mouse: structure of alternate transcripts P.J. O’Shaughnessy
a,*, P. Marsh b, K. Dudley
b
aDepartment of Veterinary Physiology, Universiry of Glasgow Veterinary School, Bearsden Rd., Glasgow G61 IQH, UK, b The Randall Institute, King’s College, London WC2B SRL, UK (Received
4 October
1993; accepted
20 December
1994)
Abstract The structure of RNA encoding the mouse ovarian follicle-stimulating hormone (FSH) receptor was studied during post-natal development and in the adult hypogonadal (hpg) mouse which lacks circulating gonadotrophins. Using reverse transcription and the polymerase chain reaction @CR) four major transcripts of the FSH receptor were found in the normal adult ovary. The largest transcript was the expected size from the position of the PCR primers (on exons 1 and 10) and sequencing confirmed that it was derived from FSH receptor mRNA. The three other transcripts were also derived from FSH receptor mRNA but they contained deletions corresponding to one or more complete exons. Each transcript lacked exon 2 while exons 5 and/or 6 were lacking in the smaller species. All four transcripts were present in ovaries of hpg mice showing that expression of receptor mRNA and development of alternate splicing are not gonadotrophin-dependent. During development in the mouse full-length FSH receptor transcripts were not detected in the ovary until day 5 although shorter transcripts were present at days 1 and 3. Results confirm that the FSH receptor primary transcript undergoes alternate splicing in the ovary and that the pattern of splicing changes as the ovary develops, probably as a result of follicular development. Key words:
FSH receptor; Ovary; Development;
(Alternate
splicing)
1. Introduction
The ovary of the newborn mouse contains largely primordial follicles, some of which develop to the antral stage during the subsequent 2 weeks (Mannan and O’Shaughnessy, 1991). Studies using the hypogonadal (hpg) mouse which lacks circulating gonadotrophins (Cattanach et al., 1977; Mason et al., 1986) have shown that the gonadotrophins are required for normal progress of follicular development during this period (Halpin et al., 1986). It remains unclear, however, at which stage of development follicles become sensitive to the gonadotrophins. Circulating levels of luteinising hormone (LH) and follicle-stimulating hormone (FSH) are high in normal neonatal mice (Stiff et al., 1974; Halpin et al., 1986) and responsiveness to these hor-
* Corresponding
author.
Elsevier Science Ireland Ltd. SSDI 0303-7207(94)00014-Z
mones will depend, therefore, upon development of functional receptors. The FSH and LH receptor genes share a number of marked similarities including a single large exon which encodes the transmembrane-spanning and cytoplasmic domains and a number of smaller exons (nine in the case of the FSH receptor) which encode most of the extracellular domain (Heckert et al., 1992). The LH receptor primary transcript has been shown to undergo alternate splicing (Loosfelt et al., 1989; Tsai-Morris et al., 1991) and in the rat ovary the appearance of functional LH receptors during development is coincidental with a change in alternate splicing of the receptor primary transcript (Sokka et al., 1992). Before 7 days of age only truncated versions of the LH-R mRNA are present while larger species appear after 7 days, the time at which functional receptors first appear (Sokka and Huhtaniemi, 1990). It is now clear that the FSH receptor primary transcript also undergoes alternate splicing although the pattern of splicing appears
198
P.J. O’Shaughnessy et al. /Molecular
and Cellular Endocrinology 101 (1994) 197-201
to differ from the LH receptor and to differ between species (Kelton et al., 1992; Gromoll et al., 1992; O’Shaughnessy and Dudley, 1993; Khan et al., 1993). In this study we have determined the structure of FSH receptor primary transcripts present in the mouse ovary and have examined expression of these transcripts during development in the normal ovary and in the adult hpg using reverse transcription and the polymerase chain reaction CRT-PCR). Results show that mRNA species containing all 10 exons of the receptor gene are first present at day 5 in the normal mouse ovary but that shorter transcripts are present at earlier ages.
2.4. Sequencing Products of PCR amplification of cDNA from normal adult ovaries were identified on agarose gels by ethidium bromide staining and individual bands were cut out and separated from the agarose by electroelution. The PCR products were sequenced directly by the dideoxy-chain termination method as described by Winfield (1989).
3. Results 3.1. FSH receptor in adult normal and hpg ovaries
2. Materials and methods 2.1. Animals
Normal and hpg mice were bred at the University of Glasgow Veterinary School as described previously (O’Shaughnessy and Dudley, 1993). Normal mice were used when adult or on days 1 (day of birth), 3, 5, 7, 10 or 15 and hpg mice were used when adult. Animals were killed by decapitation and ovaries stored in liquid N, until RNA extraction.
Total RNA from ovaries of adult normal and hpg mice was reverse transcribed and FSH-R cDNA amplified by PCR. As observed previously in the mouse testis (O’Shaughnessy and Dudley, 19931, four major PCR products were obtained ranging from the expected size of about 780 bp down to approximately 550 bp (Fig. 1, lane 1). These products have been designated mFSH-Rl to mFSH-R4. A similar pattern of PCR products was found in cDNA derived from both normal and hpg animals although the relative intensity of the bands appeared different (Fig. 1).
2.2. RT-PCR
3.2, Sequence of alternate transcripts
Total RNA was extracted from pools of 4-8 ovaries at each age or from ovaries of individual animals using the guanidinium thiocyanate/acid phenol method (Chomczynski and Sacchi, 1987). RNA was reverse transcribed using random hexamers and M-MLV reverse transcriptase (Stratagene Ltd. Cambridge, UK) and this cDNA was used as a template for PCR (O’Shaughnessy and Murphy, 1993). The primers used for PCR amplification of the FSH receptor were: (a) 5’ TCCCCCGGAACGCCATTGAA 3’, (b) 5’ GCCTTAAAATAGACTTGTTGCA 3’. These primers are based on the rat FSH-R cDNA sequence (Sprengel et al., 1990; O’Shaughnessy and Dudley, 1993) and are situated on the first and last exons (1 and 10) to give an expected product size of 779 bp. As an internal standard p-actin mRNA was reverse transcribed and amplified by PCR (O’Shaughnessy and Murphy, 1993).
To determine the structure of these PCR products each band was isolated and sequenced (Fig. 2). The sequence of mFSH-Rl confirmed that it was derived from FSH receptor mRNA. As expected from the position of the PCR primers the amplified product contained part of exon 1, the whole of exons 2-9 and part of exon 10. Including the complete sequence of previously exon 1, which has been published
2.3. Blotting For Southern hybridisation of PCR products the DNA was transferred from agarose gels to nitrocellulose membranes and hybridised with a 32P-labelled cDNA probe prepared from the extracellular portion of the rat FSH receptor (O’Shaughnessy and Dudley, 1993).
bp
955 7369 585 476 4
Lane
1
2
3
Fig. 1. Southern blot hybridisation of PCR products using FSH receptor primers. cDNA for amplification was prepared from adult normal ovaries (lane 1) or hpg ovaries (lane 2). Lane 3 was a PCR blank control and the position of molecular size standards is shown.
P.J. O’Shaughnessy et al. /Molecular
and Cellular Endocrinology 101 (1994) 197-201
Mouse Rat Human Sheep
FSH-R FSH-R FSH-R FSH-R
199
MALLLVSLLAFLGSGSGCHHWLCHCSNRVFLCQDSKVTEI 40 ________--___T_-----____._----------____ _________-__SL______RI___.______E---_-----_--F--A---__SL_-----R.__--G_-G----------_M
PPDLPRNAIEL
IEISQNDVLEVIEADV
90
_~_______________--__Q__~___.__--____..____~-----_~____~-“_____________~_A___------______-_-------~-
FSNLPNLHEIRIEKANNLLYINPEAFQNLPSLRYL-----K___----______~-----._..___-_----______._~-"--
140
-----K--____-----____T-__----N-Q-__-----------~~__ -----K--_____----____D-D_-----N-_--__---------~__
190
240 NGTQLDELNLSDNNNLEELPDDVFQGASGPWLDISRTKVYSLPNHGLEN --------_____-__~___N_____-_--I---_-_______~__----------__AV---------__~----H_-----I---_RIH----R*H---SY--__ ------___----S---___~----------I_-__---R~R---SY-___ LKKLRARSTYRLKKLPSLDKFVMLIEASLTYPSHCCAFANWRRQTSELH _-------________N_____T_M_-----_--___LK__~---_-------__N_____T_~_L_A-M_____----__._____.__~---______K___H_____--~___T-M______________________D-_
289
Fig. 3. Comparison between mouse, rat, sheep and human FSH receptor sequence coded by exons 1-9. Dashes indicate homology between sequences. The sequences underlined are coded for by exons deleted in alternate transcripts described in Fig. 2.
extracted from the ovaries from animals aged 1-15 days, reverse transcribed and the cDNA amplified by PCR. On days 5, 7, 10 and 15 bands corresponding to the four different FSH receptor alternate transcripts were apparent on Southern blotting (Fig. 4). Also present on days 7-15 were one or two smaller bands
Fig. 2. Sequence of four FSH receptor transcripts isolated following PCR amplification of cDNA from adult normal ovaries. JZxon 1 is assumed to be common to all transcripts and deviations from exon 2 to exon 7 are shown for transcripts l-4. Sequences in all four transcripts were identical from exon 7 to the start of exon 10. The start of each exon is indicated with a bold letter. The underlined sequence in exon 1 is from Huhtaniemi et al. (1992). Dashes indicate missing nucleotides.
(Huhtaniemi et al., 19921, homology of the primary structure encoded by exons l-9 of the mouse FSH receptor was 94, 88 and 87% with the rat (Sprengel et al., 19901, human (Minegish et al., 1991) and ovine (Yarney et al., 1993) FSH receptors respectively (Fig. 3). The sequences of the three smaller PCR products (FSH-R2, R3 and R4) confirmed that they are derived from the FSH receptor primary transcript but that they contain deletions corresponding to one or more complete exons (Fig. 2). All three alternate transcripts had exon 2 deleted while FSH-R3 had exon 5 deleted and FSH-R4 had exons 5 and 6 deleted. 3.3. FSH receptor during ovarian development To determine the stage of ovarian development at which FSH-R mRNA is first detectable RNA was
bp 955
-
736 565 476 -
Age (days)
1
Age (days)
1
3
5
7
10
15
b)
3
5
7
10
15
Fig. 4. PCR amplifications of cDNA from ovaries of different ages. (a) Southern blot hybridisation following PCR amplification using FSH receptor primers. The template for PCR was prepared from animals aged l-15 days as shown. The position of molecular size standards is indicated on the left. (b) Ethidium bromide staining of p-actin PCR products from the same cDNA template used in a.
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et al. /Molecular
and Cellular Endocrinology
bp 736 585476 -
blank
2 animal
1
Fig. 5. Southern blot hybridisation of PCR products from ovaries of two newborn (day 1) mice using FSH receptor primers. Lane 1 was a blank control and template for lanes 2 and 3 was prepared from individual animals as shown.
which may represent further alternate transcripts although these have not been characterised. On days 1 and 3 no full-length transcripts were detectable with only FSH-R2 and R4 present on day 1 and FSH-R3 and R4 present on day 3. Further experiments using ovaries of individual newborn animals, reverse transcribed and amplified by PCR, confirmed that only shorter transcripts are present on day 1 (Fig. 5) although the predominant transcript varied between animals with R2 present in animal 1 and R2 and R3 present in animal 2.
4. Discussion From previous studies and results reported here it is clear that receptors for both gonadotrophins show alternate splicing although the pattern of splicing between gonadotrophin receptors appears to differ. Assuming that the structural organisation of the mouse FSH receptor gene resembles that of the rat gene the alternate transcripts described here lack exons 2, 5 and/or 6. Since the intron phase is 2 for all introns of the rat FSH receptor gene this does not affect the reading frame of the transcripts. Each of the alternate transcripts contains the first and last exons making it possible that they are translated although we have no evidence for this. Since the last exon encodes the membrane-spanning and intracellular regions it is also possible that the resultant proteins are functional. It has been reported that amino acids 9-30 are involved in binding of FSH to its receptor (Dattatreyamurty and Reichert, 1992). These amino acids are coded for by exon 1 which is present in all three alternate transcripts suggesting that they may retain some hormone binding capacity. Other work, using chimeras of rat FSH and LH receptors, has shown that amino acids 18-165 (contained within exons l-6) appear to be required for normal FSH activation of cyclic AMP
101 (1994) 197-201
production (Braun et al., 1991). It is possible, therefore, that the shortened proteins, if formed, may retain some hormone binding capacity but have an altered ability to stimulate second messenger production. Previous studies have shown the presence of two alternate FSH receptor transcripts in the human testis (Gromoll et al., 1992; Heckert et al., 1992) and two in the ram testis (Khan et al., 1993). The alternative transcripts in the human contain either a 251-bp insert or a deletion of exon 9 while the ovine transcripts are markedly shortened containing only exons l-4 or 1-8 of the FSH receptor full-length transcript. Each of the transcripts reported here differs, therefore, from those described in other species. It is uncertain whether the alternate transcripts described in human and sheep are also present in the mouse. All of the transcripts reported here contained exon 9 and if any other transcripts exist in the mouse without exon 9 they must be relatively minor. The PCR conditions used in experiments described here were such that transcripts containing only exons l-4 or l-8 would not be amplified and these species may exist in the mouse. The pattern of alternate splicing in the mouse ovary closely resembles that in the mouse testis (O’Shaughnessy and Dudley, 1993) and, despite the lack of sequence information on the testicular transcripts, suggests that there is no major sex difference in processing of the FSH receptor primary transcript. In the ovary, FSH receptors are present only on the granulosa cells (Zeleznik et al., 1974; Uilenbroek and van der Linden, 1983) and changes in receptor number or receptor splicing must reflect changes in granulosa cell populations as the ovary develops. Transcripts of the FSH-R were present in the mouse ovary at birth although the predominant transcript present in the neonates varied between animals. At birth only primordial follicles with an incomplete granulosa cell layer (type 2 follicles) are present in the mouse ovary (Mannan and O’Shaughnessy, 1991) but type 3 primordial follicles (complete granulosa cell ring) are present at day 3 showing that follicular development is occurring at birth. Splicing is normally very precisely controlled and variations may reflect differences in the degree of follicle development between animals. Despite the variation in younger animals full-length receptor mRNA transcripts did not appear until day 5 at which time the ovary contains numerous primary follicles. In studies by Fortune and Eppig (1979) ovaries from 2-day-old mice did not respond to FSH in culture whereas ovaries from 7-day-old mice responded strongly. Similarly, Sokka and Huhtaniemi (1990) first observed an acute, FSH-sensitive, cyclic AMP response in ovaries from rats aged 4 days (the morphology of rat and mouse ovaries is similar at this stage suggesting that comparison between the species is valid (Funkenstein et al., 1980)). This is the time period during which
P.J. O’Shaughnessy et al. /Molecular
and Cellular Endocrinology 101 (1994) 197-201
full-length FSH-R mRNA first appears and would be consistent with the full-length receptors having, at least, greater activity than the shorter species. Terada et al. (1984) have reported, however, that ovaries from foetal and neonatal mice will respond to FSH when cultured for 48 h. This might suggest that the shorter mRNA transcripts encode for functional receptors although what effect the culture period might have on the splicing pattern is unknown. The presence of FSH receptor transcripts in the neonatal ovary does confirm, however, that the follicular cells of the primordial follicle show differentiated function usually associated with more developed granulosa cells. The hpg mouse has a deletion in the gene encoding gonadotrophin-releasing hormone (Mason et al., 1986). As a consequence, the circulating levels of FSH and LH are undetectable and the gonads develop in an environment which is essentially gonadotrophin-free (Cattanach et al., 1977). The ovaries of these hpg mice contained FSH-R mRNA and all four major transcripts were present demonstrating that the gonadotrophins are not required for expression of the FSH receptor gene or for the appearance of full-length transcripts. The adult hpg ovary contains follicles up to the late secondary stage (more than one layer of granulosa cells but no follicular fluid) which is similar to ovaries of normal mice aged 5-7 days (Halpin et al., 1986; Mannan and O’Shaughnessy, 19911. This suggests that the splicing pattern of FSH-R mRNA depends on the stage of follicular development although the gonadotrophins may have a direct effect on the relative amounts of each transcript. It has been proposed that elevated levels of circulating FSH may be required during the neonatal period for subsequent development of FSH receptors (Smith and Ojeda, 1986). Others, however, have found no requirement for elevated FSH during this period (Sokka and Huhtaniemi, 1990). Results using the hpg mouse tend to support the latter view that gonadotrophins are not required although quantitative studies have not been carried out to determine whether receptor protein and mRNA levels are normal.
5. Acknowledgements This study was supported by the AFRC. We should like to thank Elizabeth Dunlop for technical assistance.
201
6. References Braun, T., Schofield, P.R. and Sprengel, R. (1991) EMBO J. 10, 1885-1890. Cattanach. B.M., Iddon, C.A., Charlton, H.M.. Chiappa. S.A. and Fink, G. (1977) Nature 269, 338-340. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. Dattatreyamurty, B. and Reichert, L.E. (1992) Mol. Cell. Endocrinol. 87, 9-17. Fortune. J.E. and Eppig, J.J. (1979) Endocrinology 105, 760-768. Funkenstein, Nimrod. A. and Lindner, H.R. (1980) Endocrinology 106, 98-106. Gromoll, J., Gudermann, T. and Nieschlag, E. (1992) Biochem. Biophys. Res. Commun. 188, 1077-1083. Halpin, D.M., Jones, A., Fink, G. and Charlton, H.M. (1986) J. Reprod. Fertil. 77, 287-296. Heckert, L.L., Daley, I.J. and Griswold, M.D. (1991) Mol. Endocrinol. 6, 70-80. Kelton, CA., Cheng, S.V.Y., Nugent, N.P., Schweickhardt, R.L.. Rosenthal, J.L., Overton, S.A., Wands, G.D., Kuzeja, J.B., Luchette, CA. and Chappel. S.C. (1992) Mol. Cell. Endocrinol. 89. 141-151. Khan, H., Yarney, T.A. and Sairam. M.R. (1993) Biochem. Biophys. Res. Commun. 190, 888-894. Loosfelt, H., Misrahi, M.. Atger, R.S., Thi, M.T.V.H., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Garnier, J. and Milgrom, E. (1989) Science 245, 525-528. Mannan, M.A. and O’Shaughnessy, P.J. (1991) J. Endocrinol. 130, 101-106. Mason, A.J., Hayflick, J.S., Zoeller, R.T., Young, W.S., Phillips, H.S., Nikolics, K. and Seeburg, T.A. (1986) Science 234, 13661371. Minegish, T., Nakamura, K., Takakura, Y., Ibuki, Y. and Igarashi, M. (1991) Biochem. Biophys. Res. Commun. 175, 1125-1130. O’Shaughnessy, P.J. and Dudley, K. (1993) J. Mol. Endocrinol. 10, 363-366. O’Shaughnessy, P.J. and Murphy, L. (1993) J. Mol. Endocrinol. 11, 77-82. Smith, S.S. and Ojeda, S.R. (1986) Biol. Reprod. 34. 219-227. Sokka. T. and Huhtaniemi, I. (1990) J. Endocrinol. 127, 297-303. Sokka, T., Hamalainen, T. and Huhtaniemi, I. (1992) Endocrinology 130, 1738-1740. Sprengel, R., Braun, T., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1990) Mol. Endocrinol. 4, 525-530. Stiff, M.E., Bronson, F.H. and Stetson, M.H. (1974) Endocrinology 94, 492-496. Terada, N., Kuroda, H., Namiki, M., Kitamura, Y. and Matsumoto, K. (1984) J. Steroid Biochem. 20, 741-745. Tsai-Morris, C.H., Buczo, E., Wang. W., Xie, X.-Z. and Dufau, M.L. (1991) J. Biol. Chem. 266, 11355-11359. Uilenbroek, J.Th.J. and van der Linden, R. (1983) Acta Endocrinol. 103,413-419. Winfield, P.R. (1989) Nucleic Acids Res. 17, 1266. Yarney. T.A., Sairam, M.R., Khan, H., Ravindranath, N., Payne, S. and Seidah, N.G. (1993) Mol. Cell. Endocrinol. 93, 219-226. Zeleznik, A.J.. Midgley, A.R. and Reichert. L.E. (1974) Endocrinology 95, 818-825.
and
Cellular
Endocrinology
ELSEVIER
Molecular
and Cellular
Endocrinology
101 (1994) 197-201
Follicle-stimulating hormone receptor mRNA in the mouse ovary during post-natal development in the normal mouse and in the adult hypogonadal ( hpg) mouse: structure of alternate transcripts P.J. O’Shaughnessy
a,*, P. Marsh b, K. Dudley
b
aDepartment of Veterinary Physiology, Universiry of Glasgow Veterinary School, Bearsden Rd., Glasgow G61 IQH, UK, b The Randall Institute, King’s College, London WC2B SRL, UK (Received
4 October
1993; accepted
20 December
1994)
Abstract The structure of RNA encoding the mouse ovarian follicle-stimulating hormone (FSH) receptor was studied during post-natal development and in the adult hypogonadal (hpg) mouse which lacks circulating gonadotrophins. Using reverse transcription and the polymerase chain reaction @CR) four major transcripts of the FSH receptor were found in the normal adult ovary. The largest transcript was the expected size from the position of the PCR primers (on exons 1 and 10) and sequencing confirmed that it was derived from FSH receptor mRNA. The three other transcripts were also derived from FSH receptor mRNA but they contained deletions corresponding to one or more complete exons. Each transcript lacked exon 2 while exons 5 and/or 6 were lacking in the smaller species. All four transcripts were present in ovaries of hpg mice showing that expression of receptor mRNA and development of alternate splicing are not gonadotrophin-dependent. During development in the mouse full-length FSH receptor transcripts were not detected in the ovary until day 5 although shorter transcripts were present at days 1 and 3. Results confirm that the FSH receptor primary transcript undergoes alternate splicing in the ovary and that the pattern of splicing changes as the ovary develops, probably as a result of follicular development. Key words:
FSH receptor; Ovary; Development;
(Alternate
splicing)
1. Introduction
The ovary of the newborn mouse contains largely primordial follicles, some of which develop to the antral stage during the subsequent 2 weeks (Mannan and O’Shaughnessy, 1991). Studies using the hypogonadal (hpg) mouse which lacks circulating gonadotrophins (Cattanach et al., 1977; Mason et al., 1986) have shown that the gonadotrophins are required for normal progress of follicular development during this period (Halpin et al., 1986). It remains unclear, however, at which stage of development follicles become sensitive to the gonadotrophins. Circulating levels of luteinising hormone (LH) and follicle-stimulating hormone (FSH) are high in normal neonatal mice (Stiff et al., 1974; Halpin et al., 1986) and responsiveness to these hor-
* Corresponding
author.
Elsevier Science Ireland Ltd. SSDI 0303-7207(94)00014-Z
mones will depend, therefore, upon development of functional receptors. The FSH and LH receptor genes share a number of marked similarities including a single large exon which encodes the transmembrane-spanning and cytoplasmic domains and a number of smaller exons (nine in the case of the FSH receptor) which encode most of the extracellular domain (Heckert et al., 1992). The LH receptor primary transcript has been shown to undergo alternate splicing (Loosfelt et al., 1989; Tsai-Morris et al., 1991) and in the rat ovary the appearance of functional LH receptors during development is coincidental with a change in alternate splicing of the receptor primary transcript (Sokka et al., 1992). Before 7 days of age only truncated versions of the LH-R mRNA are present while larger species appear after 7 days, the time at which functional receptors first appear (Sokka and Huhtaniemi, 1990). It is now clear that the FSH receptor primary transcript also undergoes alternate splicing although the pattern of splicing appears
198
P.J. O’Shaughnessy et al. /Molecular
and Cellular Endocrinology 101 (1994) 197-201
to differ from the LH receptor and to differ between species (Kelton et al., 1992; Gromoll et al., 1992; O’Shaughnessy and Dudley, 1993; Khan et al., 1993). In this study we have determined the structure of FSH receptor primary transcripts present in the mouse ovary and have examined expression of these transcripts during development in the normal ovary and in the adult hpg using reverse transcription and the polymerase chain reaction CRT-PCR). Results show that mRNA species containing all 10 exons of the receptor gene are first present at day 5 in the normal mouse ovary but that shorter transcripts are present at earlier ages.
2.4. Sequencing Products of PCR amplification of cDNA from normal adult ovaries were identified on agarose gels by ethidium bromide staining and individual bands were cut out and separated from the agarose by electroelution. The PCR products were sequenced directly by the dideoxy-chain termination method as described by Winfield (1989).
3. Results 3.1. FSH receptor in adult normal and hpg ovaries
2. Materials and methods 2.1. Animals
Normal and hpg mice were bred at the University of Glasgow Veterinary School as described previously (O’Shaughnessy and Dudley, 1993). Normal mice were used when adult or on days 1 (day of birth), 3, 5, 7, 10 or 15 and hpg mice were used when adult. Animals were killed by decapitation and ovaries stored in liquid N, until RNA extraction.
Total RNA from ovaries of adult normal and hpg mice was reverse transcribed and FSH-R cDNA amplified by PCR. As observed previously in the mouse testis (O’Shaughnessy and Dudley, 19931, four major PCR products were obtained ranging from the expected size of about 780 bp down to approximately 550 bp (Fig. 1, lane 1). These products have been designated mFSH-Rl to mFSH-R4. A similar pattern of PCR products was found in cDNA derived from both normal and hpg animals although the relative intensity of the bands appeared different (Fig. 1).
2.2. RT-PCR
3.2, Sequence of alternate transcripts
Total RNA was extracted from pools of 4-8 ovaries at each age or from ovaries of individual animals using the guanidinium thiocyanate/acid phenol method (Chomczynski and Sacchi, 1987). RNA was reverse transcribed using random hexamers and M-MLV reverse transcriptase (Stratagene Ltd. Cambridge, UK) and this cDNA was used as a template for PCR (O’Shaughnessy and Murphy, 1993). The primers used for PCR amplification of the FSH receptor were: (a) 5’ TCCCCCGGAACGCCATTGAA 3’, (b) 5’ GCCTTAAAATAGACTTGTTGCA 3’. These primers are based on the rat FSH-R cDNA sequence (Sprengel et al., 1990; O’Shaughnessy and Dudley, 1993) and are situated on the first and last exons (1 and 10) to give an expected product size of 779 bp. As an internal standard p-actin mRNA was reverse transcribed and amplified by PCR (O’Shaughnessy and Murphy, 1993).
To determine the structure of these PCR products each band was isolated and sequenced (Fig. 2). The sequence of mFSH-Rl confirmed that it was derived from FSH receptor mRNA. As expected from the position of the PCR primers the amplified product contained part of exon 1, the whole of exons 2-9 and part of exon 10. Including the complete sequence of previously exon 1, which has been published
2.3. Blotting For Southern hybridisation of PCR products the DNA was transferred from agarose gels to nitrocellulose membranes and hybridised with a 32P-labelled cDNA probe prepared from the extracellular portion of the rat FSH receptor (O’Shaughnessy and Dudley, 1993).
bp
955 7369 585 476 4
Lane
1
2
3
Fig. 1. Southern blot hybridisation of PCR products using FSH receptor primers. cDNA for amplification was prepared from adult normal ovaries (lane 1) or hpg ovaries (lane 2). Lane 3 was a PCR blank control and the position of molecular size standards is shown.
P.J. O’Shaughnessy et al. /Molecular
and Cellular Endocrinology 101 (1994) 197-201
Mouse Rat Human Sheep
FSH-R FSH-R FSH-R FSH-R
199
MALLLVSLLAFLGSGSGCHHWLCHCSNRVFLCQDSKVTEI 40 ________--___T_-----____._----------____ _________-__SL______RI___.______E---_-----_--F--A---__SL_-----R.__--G_-G----------_M
PPDLPRNAIEL
IEISQNDVLEVIEADV
90
_~_______________--__Q__~___.__--____..____~-----_~____~-“_____________~_A___------______-_-------~-
FSNLPNLHEIRIEKANNLLYINPEAFQNLPSLRYL-----K___----______~-----._..___-_----______._~-"--
140
-----K--____-----____T-__----N-Q-__-----------~~__ -----K--_____----____D-D_-----N-_--__---------~__
190
240 NGTQLDELNLSDNNNLEELPDDVFQGASGPWLDISRTKVYSLPNHGLEN --------_____-__~___N_____-_--I---_-_______~__----------__AV---------__~----H_-----I---_RIH----R*H---SY--__ ------___----S---___~----------I_-__---R~R---SY-___ LKKLRARSTYRLKKLPSLDKFVMLIEASLTYPSHCCAFANWRRQTSELH _-------________N_____T_M_-----_--___LK__~---_-------__N_____T_~_L_A-M_____----__._____.__~---______K___H_____--~___T-M______________________D-_
289
Fig. 3. Comparison between mouse, rat, sheep and human FSH receptor sequence coded by exons 1-9. Dashes indicate homology between sequences. The sequences underlined are coded for by exons deleted in alternate transcripts described in Fig. 2.
extracted from the ovaries from animals aged 1-15 days, reverse transcribed and the cDNA amplified by PCR. On days 5, 7, 10 and 15 bands corresponding to the four different FSH receptor alternate transcripts were apparent on Southern blotting (Fig. 4). Also present on days 7-15 were one or two smaller bands
Fig. 2. Sequence of four FSH receptor transcripts isolated following PCR amplification of cDNA from adult normal ovaries. JZxon 1 is assumed to be common to all transcripts and deviations from exon 2 to exon 7 are shown for transcripts l-4. Sequences in all four transcripts were identical from exon 7 to the start of exon 10. The start of each exon is indicated with a bold letter. The underlined sequence in exon 1 is from Huhtaniemi et al. (1992). Dashes indicate missing nucleotides.
(Huhtaniemi et al., 19921, homology of the primary structure encoded by exons l-9 of the mouse FSH receptor was 94, 88 and 87% with the rat (Sprengel et al., 19901, human (Minegish et al., 1991) and ovine (Yarney et al., 1993) FSH receptors respectively (Fig. 3). The sequences of the three smaller PCR products (FSH-R2, R3 and R4) confirmed that they are derived from the FSH receptor primary transcript but that they contain deletions corresponding to one or more complete exons (Fig. 2). All three alternate transcripts had exon 2 deleted while FSH-R3 had exon 5 deleted and FSH-R4 had exons 5 and 6 deleted. 3.3. FSH receptor during ovarian development To determine the stage of ovarian development at which FSH-R mRNA is first detectable RNA was
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Fig. 4. PCR amplifications of cDNA from ovaries of different ages. (a) Southern blot hybridisation following PCR amplification using FSH receptor primers. The template for PCR was prepared from animals aged l-15 days as shown. The position of molecular size standards is indicated on the left. (b) Ethidium bromide staining of p-actin PCR products from the same cDNA template used in a.
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Fig. 5. Southern blot hybridisation of PCR products from ovaries of two newborn (day 1) mice using FSH receptor primers. Lane 1 was a blank control and template for lanes 2 and 3 was prepared from individual animals as shown.
which may represent further alternate transcripts although these have not been characterised. On days 1 and 3 no full-length transcripts were detectable with only FSH-R2 and R4 present on day 1 and FSH-R3 and R4 present on day 3. Further experiments using ovaries of individual newborn animals, reverse transcribed and amplified by PCR, confirmed that only shorter transcripts are present on day 1 (Fig. 5) although the predominant transcript varied between animals with R2 present in animal 1 and R2 and R3 present in animal 2.
4. Discussion From previous studies and results reported here it is clear that receptors for both gonadotrophins show alternate splicing although the pattern of splicing between gonadotrophin receptors appears to differ. Assuming that the structural organisation of the mouse FSH receptor gene resembles that of the rat gene the alternate transcripts described here lack exons 2, 5 and/or 6. Since the intron phase is 2 for all introns of the rat FSH receptor gene this does not affect the reading frame of the transcripts. Each of the alternate transcripts contains the first and last exons making it possible that they are translated although we have no evidence for this. Since the last exon encodes the membrane-spanning and intracellular regions it is also possible that the resultant proteins are functional. It has been reported that amino acids 9-30 are involved in binding of FSH to its receptor (Dattatreyamurty and Reichert, 1992). These amino acids are coded for by exon 1 which is present in all three alternate transcripts suggesting that they may retain some hormone binding capacity. Other work, using chimeras of rat FSH and LH receptors, has shown that amino acids 18-165 (contained within exons l-6) appear to be required for normal FSH activation of cyclic AMP
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production (Braun et al., 1991). It is possible, therefore, that the shortened proteins, if formed, may retain some hormone binding capacity but have an altered ability to stimulate second messenger production. Previous studies have shown the presence of two alternate FSH receptor transcripts in the human testis (Gromoll et al., 1992; Heckert et al., 1992) and two in the ram testis (Khan et al., 1993). The alternative transcripts in the human contain either a 251-bp insert or a deletion of exon 9 while the ovine transcripts are markedly shortened containing only exons l-4 or 1-8 of the FSH receptor full-length transcript. Each of the transcripts reported here differs, therefore, from those described in other species. It is uncertain whether the alternate transcripts described in human and sheep are also present in the mouse. All of the transcripts reported here contained exon 9 and if any other transcripts exist in the mouse without exon 9 they must be relatively minor. The PCR conditions used in experiments described here were such that transcripts containing only exons l-4 or l-8 would not be amplified and these species may exist in the mouse. The pattern of alternate splicing in the mouse ovary closely resembles that in the mouse testis (O’Shaughnessy and Dudley, 1993) and, despite the lack of sequence information on the testicular transcripts, suggests that there is no major sex difference in processing of the FSH receptor primary transcript. In the ovary, FSH receptors are present only on the granulosa cells (Zeleznik et al., 1974; Uilenbroek and van der Linden, 1983) and changes in receptor number or receptor splicing must reflect changes in granulosa cell populations as the ovary develops. Transcripts of the FSH-R were present in the mouse ovary at birth although the predominant transcript present in the neonates varied between animals. At birth only primordial follicles with an incomplete granulosa cell layer (type 2 follicles) are present in the mouse ovary (Mannan and O’Shaughnessy, 1991) but type 3 primordial follicles (complete granulosa cell ring) are present at day 3 showing that follicular development is occurring at birth. Splicing is normally very precisely controlled and variations may reflect differences in the degree of follicle development between animals. Despite the variation in younger animals full-length receptor mRNA transcripts did not appear until day 5 at which time the ovary contains numerous primary follicles. In studies by Fortune and Eppig (1979) ovaries from 2-day-old mice did not respond to FSH in culture whereas ovaries from 7-day-old mice responded strongly. Similarly, Sokka and Huhtaniemi (1990) first observed an acute, FSH-sensitive, cyclic AMP response in ovaries from rats aged 4 days (the morphology of rat and mouse ovaries is similar at this stage suggesting that comparison between the species is valid (Funkenstein et al., 1980)). This is the time period during which
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full-length FSH-R mRNA first appears and would be consistent with the full-length receptors having, at least, greater activity than the shorter species. Terada et al. (1984) have reported, however, that ovaries from foetal and neonatal mice will respond to FSH when cultured for 48 h. This might suggest that the shorter mRNA transcripts encode for functional receptors although what effect the culture period might have on the splicing pattern is unknown. The presence of FSH receptor transcripts in the neonatal ovary does confirm, however, that the follicular cells of the primordial follicle show differentiated function usually associated with more developed granulosa cells. The hpg mouse has a deletion in the gene encoding gonadotrophin-releasing hormone (Mason et al., 1986). As a consequence, the circulating levels of FSH and LH are undetectable and the gonads develop in an environment which is essentially gonadotrophin-free (Cattanach et al., 1977). The ovaries of these hpg mice contained FSH-R mRNA and all four major transcripts were present demonstrating that the gonadotrophins are not required for expression of the FSH receptor gene or for the appearance of full-length transcripts. The adult hpg ovary contains follicles up to the late secondary stage (more than one layer of granulosa cells but no follicular fluid) which is similar to ovaries of normal mice aged 5-7 days (Halpin et al., 1986; Mannan and O’Shaughnessy, 19911. This suggests that the splicing pattern of FSH-R mRNA depends on the stage of follicular development although the gonadotrophins may have a direct effect on the relative amounts of each transcript. It has been proposed that elevated levels of circulating FSH may be required during the neonatal period for subsequent development of FSH receptors (Smith and Ojeda, 1986). Others, however, have found no requirement for elevated FSH during this period (Sokka and Huhtaniemi, 1990). Results using the hpg mouse tend to support the latter view that gonadotrophins are not required although quantitative studies have not been carried out to determine whether receptor protein and mRNA levels are normal.
5. Acknowledgements This study was supported by the AFRC. We should like to thank Elizabeth Dunlop for technical assistance.
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