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1580

BIOLOGY OF REPRODUCTION 70, 1580–1588 (2004)
Published online before print 28 January 2004.
DOI 10.1095/biolreprod.103.026898
Downregulation of Follicle-Stimulating Hormone (FSH)-Receptor Messenger RNA
Levels in the Hamster Ovary: Effect of the Endogenous and Exogenous FSH
1
Yi-Ming Zhang
3
and Shyamal K. Roy
2,3,4
Departments of Physiology and Biophysics
3
and Obstetrics and Gynecology,
4
University of Nebraska Medical Center,
Omaha, Nebraska 68198-4515
ABSTRACT
Although gonadotropins have been reported to downregulate
FSH-receptor (FSHR) mRNA levels in the ovaries of female rats,
the effect of the gonadotropin surge, particularly FSH, on ham-
ster follicular FSHR mRNA levels warrants further examination.
The objectives of the present study were to clone and determine
the complete FSHR cDNA sequence of the hamster and to de-
lineate the effects of endogenous and exogenous FSH on the
steady-state levels of ovarian FSHR mRNA. Complete FSHR
cDNA was derived from hamster ovarian total RNA by the strat-
egy of 3؅- and 5؅-rapid amplification of cDNA ends. Ovaries
were obtained before and after the endogenous gonadotropin
surge or exogenous FSH administration, and the steady-state lev-
els of FSHR mRNA were assessed by Northern blot hybridiza-
tion. Cloned FSHR cDNA consists of a reading frame corre-
sponding to exons 1–10 of the human FSHR gene and the 5؅-
and 3؅-untranslated regions. The nucleic acid and amino acid
sequences of the reading frame were at least 87% and 92%
identical, respectively, to that of human, rat, and mouse FSHR.
Furthermore, the amino acid sequence contained seven trans-
membrane domains characteristic of the FSHR. The steady-state
levels of FSHR mRNA increased from estrus (Day 1) to reach a
peak on proestrus (Day 4) noon; however, significant attenua-
tion was noted following the gonadotropin surge, which was
blocked by phenobarbital. Exogenous FSH also downregulated,
both dose- and time-dependently, ovarian FSHR mRNA levels.
These data indicate that the nucleic acid sequence of hamster
FSHR has been identified and that FSH modulates FSHR mRNA
levels in the hamster ovary.
follicle-stimulating hormone, follicle-stimulating hormone recep-
tor, granulosa cells, ovary
INTRODUCTION
Ovarian follicular development depends on FSH action
[1]. Lack of FSH function because of a null mutation in
the FSH␤ [2], or FSH-receptor (FSHR) [3] gene, or hy-
pophysectomy [1] in rodents results in impaired follicular
development, leading to ovulation failure and infertility.
Similarly, neutralization of serum FSH by an anti-FSH se-
rum results in cessation of primordial follicle formation in
1
Supported by grants HD28165 and HD38468 from the National Institute
of Child Health and Human Development (NICHHD, NIH) to S.K.R. The
full-length cDNA sequence of the hamster FSH receptor has been depos-
ited in the GenBank (accession no. AY509907).
2
Correspondence: Shyamal K. Roy, Departments of OB/GYN and Physi-
ology and Biophysics, DRC 5013, University of Nebraska Medical Center,
Omaha, NE 68198-4515. FAX: 402 559 6164; e-mail: skroy@unmc.edu
Received: 22 December 2003.
First decision: 20 January 2004.
Accepted: 22 January 2004.
ᮊ 2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
neonatal hamsters [4]. The action of FSH is mediated by
its cognate receptor, which belongs to a G protein-coupled
receptor superfamily. The FSHRs contain a large extracel-
lular domain that binds to the ligand [5, 6]. Heckert et al.
[7] have demonstrated that the extracellular domain of rat
FSHR is encoded by nine exons, whereas the entire trans-
membrane and the C-terminal cytoplasmic domain is en-
coded by exon 10. Furthermore, these exons are separated
by nine introns [5]. Similar structural organization has been
reported for the human FSHR gene [6]. The nucleic acid
and corresponding amino acid sequences of FSHR have
been determined for many species [5, 7–9], and this infor-
mation has led to a significant understanding of the patho-
physiology of FSHR gene expression [5, 10–12]. Because
FSH has significant roles in hamster ovarian folliculoge-
nesis [1, 4, 13, 14], identifying the nucleic acid and amino
acid sequences of hamster FSHR is essential to understand-
ing the regulation of FSHR gene expression in hamster go-
nads.
Whereas high doses of FSH have been shown to down-
regulate FSHR mRNA levels both in vivo [15–17] and in
vitro [18], a small dose of FSH has been shown to maintain
FSHR mRNA levels in cultured porcine granulosa cells
[19]. Furthermore, ovulation induction in the rat with a re-
combinant human FSH leads to suppression of FSHR
mRNA levels in the granulosa cells [20]. Because granulosa
cells differentiate into LH-responsive, progesterone-produc-
ing luteal cells after the preovulatory gonadotropin surge
[21], decreases in the levels of FSHR mRNA and protein
following the surge is expected. In the hamster, serum FSH
levels decline from the day of estrus to the morning of
proestrus, followed by a significant increase at 1500 h [22]
and a surge at 1600 h in proestrus [23]. Despite this infor-
mation, it is unclear whether the in vivo FSH surge influ-
ences the levels of FSHR mRNA in the hamster ovary dur-
ing the estrous cycle. Because FSH plays very important
roles during hamster follicular development [1, 13], it will
be important to know if FSH, whether endogenous or exo-
genous, influences ovarian FSHR mRNA levels. This in-
formation will be particularly relevant to understanding
how preantral follicles grow after the preovulatory gonad-
otropin surge [24, 25]. The objectives of the present study
were to identify the complete cDNA sequence of the ham-
ster FSHR, to determine the steady-state levels of FSHR
mRNA in the ovary during the estrous cycle, and to eval-
uate the effects of endogenous and exogenous FSH on
FSHR mRNA levels.
MATERIALS AND METHODS
Materials
Adult golden female hamsters were purchased from Sasco (Madison,
WI) and maintained in a climate-controlled room with a 14L:10D photo-
1581 EFFECT OF GONADOTROPINS ON FSHR mRNA EXPRESSION
TABLE 1. Various primers used to clone hamster FSHR cDNA.
GeneRacer oligo (dT)
GeneRacer 3Ј-primer
F (forward)
R1 (reverse)
R2 (reverse)
GeneRacer 5Ј-primer
GeneRacer oligo RNA
5Ј-CTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18-3Ј
5Ј-GCTGTCAACGATACGCTACGTAACG-3Ј
5Ј-TGTCATCACTGGCTGTGTCAT-3Ј
5Ј-ATTTGGATGAAGTTCAGAGGTT-3Ј
5Ј-ATCCTTGTGGGATGACTCG-3Ј (hamster)
5Ј-CGACTGGAGCACGAGGACACTGA-3Ј
5Ј-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3Ј
period and free access to food and water according to Institutional Animal
Care and Use Committee (IACUC) and U.S. Department of Agriculture
guidelines. The use of hamsters for the present study was approved by the
IACUC. Females with at least three consecutive estrous cycles were used.
Ovine-FSH-20 and the FSH and LH RIA kits were kindly provided by
Dr. A.F. Parlow (Harbor-UCLA, Los Angeles, CA) at a subsidized price.
Phenobarbital (65 mg/ml USP) for human injection was purchased from
the University of Nebraska Medical Center pharmacy. The TRI reagent
and RNAeasy kit for RNA extraction was from MRC, Inc. (Cincinnati,
OH), and Qiagen (Valencia, CA), respectively. Nytran membrane for
Northern hybridization was from Schleicher and Schuell (Fisher Scientific
Company, Pittsburgh, PA), and 5Ј- and 3Ј-rapid amplification of cDNA
ends (RACE) kit and cDNA cloning kit (PCRII TOPO) were from Invi-
trogen (Carlsbad, CA). All other chemicals were either from Sigma Chem-
ical Company (St. Louis, MO) or Fisher Scientific.
5Ј- And 3Ј-RACE Cloning of Full-Length Hamster
FSHR cDNA
Total ovarian RNA was extracted by TRI reagent according to the
manufacturer’s protocol and as described previously [26, 27] and was
quantified by a spectrophotometer at 260 nm. The quality of RNA was
assured by two sharp ethidium bromide-stained bands of 18S and 28S
rRNA without any obvious smear. Furthermore, no larger size correspond-
ing to any putative DNA contamination was evident.
Two FSHR cDNAs, one containing a 3Ј-untranslated region (UTR)
plus a part of the poly A tail (henceforth called 3Ј-FSHR) and the other
containing a complete 5Ј-UTR (henceforth called 5Ј-FSHR), were gener-
ated using GeneRacer Kit (Invitrogen) according to the manufacturer’s
instructions. Briefly, to synthesize a single-stranded 3Ј-FSHR cDNA, 5 ␮g
of total ovarian RNA were denatured at 65ЊC for 5 min and then mixed
with 2.5 ␮M GeneRacer oligo (dT) (Table 1), 1 mM dNTPs, 40 U of
RNaseOUT, and 15 U of ThermoScript reverse transcriptase in a final
volume of 20 ␮l and incubated at 60ЊC for 60 min, followed by 5 min at
85ЊC. The RNA template was removed from the mixture by adding 2 U
of RNase H, followed by a 20-min incubation at 37ЊC. Double-stranded
3Ј-hamster FSHR cDNA was synthesized by polymerase chain reaction
(PCR) in a 50-␮l reaction mixture containing 1 ␮l of reverse transcription
product, 0.4 ␮M GeneRacer 3Ј-primer (complementary to the last 25 bases
of the GeneRacer oligo dT), 0.2 ␮g of a forward primer (F) (Table 1),
2.75 mM MgCl
2
, 0.2 mM dNTPs, and 2.5 U of Taq polymerase. After a
4-min denaturation step at 94ЊC, PCR was continued for 30 cycles, fol-
lowed by a final extension at 72ЊC for 10 min. The PCR conditions were
1.5 min at 94ЊC, 1 min at 58ЊC, and 2.5 min at 72ЊC. To synthesize a
single-stranded 5Ј-FSHR cDNA, a 5Ј-oligo (GeneRacer RNA oligo [dT])
(Table 1) was ligated to the 5Ј-cap site of all mRNA in the total RNA and
reverse transcribed in the presence of a FSHR gene-specific reverse primer
R1 (Table 1). Double-stranded 5Ј-hamster FSHR cDNA was synthesized
by PCR using R2 and GeneRacer 5Ј-primers (Table 1). The PCR condi-
tions were 4 min denaturation at 95ЊC followed by 1 min at 94ЊC, 1 min
at 58ЊC, and 1 min at 72ЊC followed by a 10 min extension at 72ЊC. The
forward (F) and reverse (R1) primers, which corresponded to 51–72 and
851–873 bases, respectively, were designed from the conserved regions of
rat (GenBank accession no. L02842) [9], mouse (accession no. AF095642)
[12], and human (accession no. AY429104) [8] FSHR sequences, whereas
the R2 primer was designed from a 800-base pair (bp) hamster FSHR
cDNA, which was generated using F and R1 primers.
The PCR products were resolved in a 1% agarose gel, and cDNAs of
predicted size were purified from the gel using Oligotex II kit (Qiagen),
inserted into either pCRIITOPO (for 3Ј-FSHR) or pCR4-TOPO (for 5Ј-
FSHR) vector (Invitrogen) and transformed in TOPO10 cells (Invitrogen).
White colonies were selected under ampicillin, and after thorough screen-
ing, a part of one white colony for each cDNA type was amplified by
PCR and the presence of the insert verified by Southern blot analysis [26].
The rest of the colony was grown overnight under ampicillin selection,
and plasmid DNA was extracted and purified using Plasmid Maxi Kit
(Qiagen) according to the manufacturer’s instructions. The nucleotide se-
quences of 3Ј- and 5Ј-FSHR cDNA fragments were determined by auto-
mated DNA sequencing (Genomics Sequencing Service; Qiagen). The se-
quences were overlapped to derive the full-length hamster FSHR cDNA,
including the 5Ј- and 3Ј-UTRs. Analyses of nucleotide sequence and pre-
dicted amino acid composition of hamster FSHR were carried out using
the Vector NTI (Invitrogen) and GCG (Accelrys, San Diego, CA) pro-
grams. To avoid PCR-derived sequence errors, positive alignment of at
least three independent sequences was requested for any given area of the
hamster FSHR cDNA.
To synthesize a hamster FSHR cDNA probe for Northern hybridiza-
tion, a 504-bp cDNA template, corresponding to 477–981 bases, was gen-
erated by PCR from the 3Ј-end hamster FSHR cDNA using F and R1
primers (Table 1), cloned into pCRIITOPO vector, plasmid linearized with
StuI restriction enzyme (New England Biolab, Beverly, MA), and used as
a template for in vitro transcription of cRNA using Riboprobe kit (Pro-
mega, Madison, WI).
Ovarian FSHR mRNA Levels Throughout
the Estrous Cycle
Ovaries were collected from adult hamsters at 0900 h on Days 1–4
(estrus through proestrus [24]) and at 1200, 1600, and 2000 h on Day 4.
For negative tissue controls, kidney, liver, and adrenal glands were also
collected. Trunk blood was collected to determine the serum levels of FSH
and LH by specific RIAs.
Effect of Endogenous FSH on Ovarian FSHR mRNA
Levels
Hamsters were injected s.c. with phenobarbital (10 mg/100 g body
weight) at 1300 h on Day 4 to block the preovulatory gonadotropin surges
[22], and ovaries were collected at 2000 h on Day 4 and at 0900 h on
Day 1.
In a parallel experiment, phenobarbitone was injected at 2000 h on
Day 4 to block the second FSH surge [23, 28], and ovaries were collected
at 0900 h on Day 1. Ovaries from untreated hamsters were collected at
2000 h on Day 4 and at 0900 h on Day 1. Trunk blood was collected to
determine the serum levels of FSH and LH by specific RIAs.
Effect of Exogenous FSH on Ovarian FSHR mRNA Levels
Hamsters were injected s.c. with 1.64, 8.2, or 16.4 IU of ovine FSH-
20 in 0.1 ml of saline containing 0.5% bovine serum albumin at 0800 h
on Day 4. A second group of hamsters received 8.2 IU of recombinant
human FSH at the same time. Ovaries were collected 4 h after the injection
and used for RNA extraction.
Based on these results, in the next experiment hamsters were injected
s.c. with 16.4 IU of ovine FSH-20 at 0800 h on Day 4, and ovaries were
collected 1, 2, 4, 6, or 12 h after the injection. Hamsters allocated to the
12-h group also received phenobarbitone (10 mg/100 g body weight) at
1300 h to block the endogenous gonadotropin surge, which otherwise
would have overlapped with the exogenous FSH and made interpretation
of the data difficult. Ovaries from untreated hamsters were collected at
0800 h (0-h treatment group) or 4 h after the vehicle injection. Ovaries
were used for RNA extraction.
For all groups, tissues were removed immediately, quickly frozen in
liquid N
2
, and stored at Ϫ80ЊC until extracted for total RNA for Northern
hybridization detection of FSHR mRNA.
Measurements of Serum FSH and LH
Serum levels of gonadotropins were determined by specific RIAs using
rat-rat RIA kits from the National Pituitary Hormone Program as described
previously [23, 25, 29]. All samples were assayed at the same time to
1582 ZHANG AND ROY
avoid any interassay variation. The coefficient of intraassay variation was
7%.
Northern Hybridization
Northern hybridization analysis of ovarian FSHR mRNA was done as
described previously [26, 30]. Briefly, total RNA from the ovaries were
extracted using RNAeasy kit (Qiagen) according to the manufacturer’s
instructions, and 5 ␮g of denatured total RNA were fractionated in 1%
agarose-formaldehyde gels, capillary transferred to Nytran membrane, ul-
traviolet cross-linked, stained with 0.02% methylene blue in 0.3 M sodium
acetate (pH 5.5) to make the 28S and 18S ribosomal RNA bands visible.
The signal intensity was digitized using a UVP bioimaging system (UVP,
Upland, CA). After destaining and prehybridization, the membrane was
hybridized for 16–18 h in 10 ml of hybridization mixture containing 2ϫ
10
7
cpm [
32
P]CTP-labeled FSHR cRNA probe, 40% formamide, 0.5ϫ
Denhardt reagent, 7% SDS, and 10% dextran at 60ЊC in a Hybaid hybrid-
ization oven (Fisher Scientific). After rinsing, the membrane was exposed
to phosphor screen, and the signal (digital light unit [DLU]) was digitized
in a Cyclone phosphorimager (Perkin-Elmer, Wellesley, MA). First, the
DLU was normalized against the time of exposure. Then, the ratio of DLU
(radioactive signal) to optical density (18S ribosomal RNA) was calculated
to correct for any variation caused by differences in sample loading. Fi-
nally, values of all experimental groups were expressed as fold-changes
relative to respective controls.
Statistical Analysis
Each experiment was repeated at least three times, and the data were
analyzed by ANOVA with the Fisher protected least-significance-difference
post-hoc test using Statview software (SAS Institute, Cary, NC). The level
of significance was set at 5%.
RESULTS
Full-Length Cloning of Hamster FSHR cDNA
Because FSH influenced hamster follicular development
from the primary stage onward and no information about
hamster FSHR cDNA was available, it was necessary to
identify the complete nucleic acid sequence of the hamster
FSHR, including the 5Ј- and 3Ј-UTRs for future examina-
tion of FSHR mRNA expression in the granulosa cells. The
5Ј-RACE strategy allowed selection of only those mRNA
molecules that had an intact 5Ј-cap structure and eliminated
all truncated mRNA and non-mRNA species, thus assuring
the identification of a complete 5Ј-UTR, which started from
the most probable transcription start site. Similarly, binding
of the oligo dT primer at the 3Ј-end of the FSHR mRNA
assured the identification of a complete 3Ј-UTR. The RACE
strategy followed by sequencing revealed a 2392-bp cDNA
corresponding to FSHR mRNA, which contained a 109-bp
5Ј-UTR upstream of the translation start site, a 2082-bp
open reading frame, a stop codon, and a 198-bp 3Ј-UTR
(Fig. 1). The open reading frame was at least 90%, 87%,
and 89% similar to human [6], rat [9], and mouse [12]
FSHR cDNA, respectively. Furthermore, the open reading
frame corresponded very well to the exon 1–10 sequences
of the human FSHR [6] sequence. The 5Ј-UTR of the ham-
ster FSHR cDNA had 85% and 80% sequence similarities
with the corresponding 5Ј-UTR sequences of the mouse
(accession no. 487570) and rat (accession no. 581117)
FSHR, respectively. The deduced amino acid sequence of
the hamster FSHR corresponded to a peptide sequence of
694 amino acids, which showed at least 92% similarity with
rat, human, and mouse FSHR amino acid sequences [6, 9,
12] (Fig. 2). Furthermore, sequence comparison showed all
the characteristics of FSHR, including a 17-amino-acid sig-
nal peptide, seven transmembrane domains, four conserved
putative sites for N-linked glycosylation, 11 cysteine resi-
dues, and other putative posttranslational modification sites
(Fig. 2 and Table 2).
Northern hybridization analysis of hamster ovarian RNA
revealed a single band of FSHR mRNA of approximately
2.5 kilobases (kb) (Fig. 3). Furthermore, as expected, no
FSHR hybridization signal was detected for the kidney, ad-
renal gland, or liver, indicating the specificity of the signal
(Fig. 3). However, no alternately spliced FSHR transcript
could be detected in the ovarian sample.
Ovarian FSHR mRNA Levels Throughout
the Estrous Cycle
The steady-state levels of FSHR mRNA were relatively
low on the morning of estrus; however, a marked increase
occurred by the morning of Day 2 (Fig. 4). Thereafter, ovar-
ian FSHR mRNA levels continued increasing, to reach a
peak level by 1200 h on Day 4 (Fig. 4). In the hamster [23,
25, 31], serum levels of LH started increasing by 1400 h
on Day 4, reaching a peak at 1600 h. The FSH surge occurs
at 1500 h on Day 4, reaching a peak at 1600 h. Coinciding
with the increase in serum levels of FSH and LH, ovarian
FSHR mRNA levels decreased noticeably by 1600 h on
Day 4 (Fig. 4), and these levels declined further by 2000 h
to reach the levels of 0900 h at Day 1 (Fig. 4).
Effect of Endogenous FSH Surge on Ovarian FSHR
mRNA Levels
To determine whether the decline in ovarian FSHR
mRNA levels in the hamster was really induced by the
preovulatory gonadotropin surge, hamsters were injected
with phenobarbitone at 1300 h to block the surge [25, 28],
and the steady-state levels of ovarian FSHR mRNA were
examined at 2000 h on Day 4 and at 0900 h on Day 1.
Serum levels of LH and FSH increased significantly by
1600 h on day 4, which was completely blocked by phe-
nobarbitone pretreatment (data not shown) [22, 25]. The
block in the hormone surge correlated well with high levels
of ovarian FSHR mRNA at 2000 h on Day 4, and the levels
remained high on Day 1, 0900 h (Fig. 5). Phenobarbitone
itself had no effect on ovarian FSHR mRNA levels (data
not shown).
Because a second FSH surge occurs in the hamster by
2200 h on Day 4 [28], it was of interest to assess whether
the persistent low levels of FSHR mRNA at 0900 h on Day
1 were caused by the second FSH surge. To achieve this
goal, the occurrence of the second FSH surge was blocked
by phenobarbitone injection at 2000 h on Day 4, and FSHR
mRNA levels in the ovary were determined at 0900 h on
Day 1. Although phenobarbitone blocked the second rise in
serum FSH (data not shown) [22], FSHR mRNA levels did
not show appreciable change by 0900 h on Day 1 (Fig. 5).
Effect of Exogenous FSH on Ovarian FSHR mRNA Levels
Because serum levels of both FSH and LH increase
during the preovulatory gonadotropin surge [1], the ratio-
nale for this experiment was to determine whether FSH
alone, dose and time dependently, could mimic the effect
of the gonadotropin surge on ovarian FSHR mRNA levels.
Because the lowest levels of endogenous FSH were pre-
sent on the morning of Day 4, when the injection was
made, and started to increase only after approximately
1400 h [23], the effect of exogenous FSH on expression
of its receptor was examined on the morning of Day 4.
Ovine FSH at a dose level of 8.2 IU reduced the levels of
FSHR mRNA to 50% of the 0800-h control value (Fig.
1583 EFFECT OF GONADOTROPINS ON FSHR mRNA EXPRESSION
FIG. 1. Nucleotide sequence of the ham-
ster FSHR cDNA showing 5Ј- and 3Ј-un-
translated regions and a single open read-
ing frame identified by 5Ј- and 3Ј-RACE
strategy. Sequence corresponding to signal
peptide and stop codon are in bold. Be-
ginning of putative exons 2–10 (based on
human FSHR exon sequences) is marked
by vertical lines and labeled Ex-2 to -10.
Exon 1 sequence starts from the nucleo-
tide Ϫ109. ϩ1, Putative translation initia-
tion site.
6). A higher dose did not show additional suppression
(Fig. 6). Furthermore, 8.2 IU of recombinant human FSH
was also able to significantly reduce FSHR mRNA levels
by 4 h, and the effect was comparable to that of a similar
dose of ovine FSH (Fig. 6), thus indicating that FSH, with-
out the presence of LH, can downregulate FSHR mRNA
levels in the hamster ovary.
To compensate for the short systemic half-life of FSH
and because a comparable effect of 8.2- and 16.4-IU doses
of FSH on FSHR mRNA levels was observed, a time-
course study was done using 16.4 IU of ovine FSH. Sig-
nificant reduction of FSHR mRNA was evident by 2 h after
the FSH administration (Fig. 7), which corresponded well
with the surge of the estrous cycle. The efficacy of FSH
suppression of FSHR mRNA was maintained up to 6 h;
however, FSHR mRNA levels showed an upward trend 12
h after the hormone administration (Fig. 7). This duration
of suppression correlated well with the duration of the ef-
fect of the endogenous increase in FSH levels, which main-
tained the decline up to the morning of Day 1.
1584 ZHANG AND ROY
FIG. 2. Deduced amino acid sequence of the hamster FSHR protein and its comparison with rat, mouse, and human FSHR amino acid sequences.
Shaded areas represent the differences in the amino acid residues across species. Signal peptide sequence is underlined, and four putative N-linked
glycosylation sites are overlined. Seven putative transmembrane domains are boxed.
TABLE 2. Putative posttranslational modification sites.
Posttranslational modification Highly probable sites (amino acid sequence number)
N-glycosylation
Sulfation
Protein kinase C phosphorylation
Casein kinase II phosphorylation
Tyrosine phosphorylation
Myristylation
191–194, 199–202, 293–296, 311–314
322–336, 327–341
26–28, 251–253, 346–348, 554–556, 595–597, 666–668
78–81, 193–196, 263–266, 313–316, 330–333, 563–566
323–329
13–18, 64–69
DISCUSSION
To our knowledge, this is the first study documenting the
complete nucleotide sequence and deduced amino acid
composition of the hamster FSHR. Although information
about the complete FSHR nucleotide sequence is available
for many species [6, 12, 32, 33], information about the
hamster FSHR nucleotide sequence has been lacking. Be-
cause FSH plays an important regulatory role in hamster
preantral folliculogenesis, regulation of FSHR mRNA lev-
els in the granulosa cells during folliculogenesis will be an
important area of study. However, to address this issue, we
first need to know the complete nucleotide sequence of the
hamster FSHR. Our data show that the nucleotide and ami-
no acid sequences of hamster FSHR are approximately 90–
92% similar to those of other species. Comparison of both
the cDNA sequence and amino acid composition of the
FSHR reveals that differences in the sequences of at least
10–15% are common across species [12], indicating that
the observed differences in the nucleotide sequence or ami-
no acid residues in the hamster FSHR are not caused by
errors introduced by PCR or sequencing. Besides, the size
of the reading frame and the length of the deduced FSHR
protein corroborate well with those reported for other spe-
cies. The presence of the 17-amino-acid N-terminal signal
peptide and seven putative transmembrane domains also
match very well with those of other species [12, 33]. Fur-
thermore, three conserved putative sites for N-linked gly-
cosylation and 11 cysteine residues in the extracellular do-
main of the hamster FSHR correspond well with those of
the rat [9], mouse [12], human [6], and other species [17,
34] and have been shown to be essential for the tertiary
conformation of the binding domain of other G protein-
1585 EFFECT OF GONADOTROPINS ON FSHR mRNA EXPRESSION
FIG. 3. Northern hybridization analysis of hamster FSHR mRNA. A)
Phosphorimage showing a [
32
P]cRNA-labeled, approximately 2.5-kb
FSHR mRNA in ovarian RNA. B) Corresponding methylene blue-stained
ribosomal RNA bands. Ad, Adrenal gland; K, kidney; L, liver; OV, ovary.
FIG. 5. Effect of phenobarbitone (phenobarb) on the relative levels of
FSHR mRNA in the hamster ovary. Days of the cycle are as follows: 1,
Estrus; 4, proestrus. Bars with the same letter are significantly (P Ͻ 0.05)
different from each other.
FIG. 4. Relative levels of FSHR mRNA in the hamster ovary throughout
the estrous cycle. Days of the cycle are as follows: 1, Estrus; 2, metestrus;
3, diestrus; 4, proestrus. Bars with the same letter are significantly (P Ͻ
0.05) different from each other.
FIG. 6. Relative levels of FSHR mRNA in the hamster ovary on Day 4
following in vivo administration of increasing doses of FSH. Saline or FSH
was injected at 0800 h, and ovaries were collected 4 h later. Bars with
same letter are significantly (P Ͻ 0.05) different from each other. oFSH,
ovine FSH; rhFSH, recombinant human FSH.
coupled receptors, such as LH [35]. However, in contrast
to other rodent species, hamster FSHR contains one more
putative N-linked glycosylation site at positions 311–314 in
the extracellular domain. The presence of a fourth N-linked
glycosylation site in the extracellular domain has been re-
ported for human and monkey FSHR [17]. Therefore, ham-
ster FSHR seems to be structurally similar to the primate
and human FSHR.
This pattern of glycosylation appears to be important for
proper folding and membrane trafficking of the FSHR [36].
Besides these, domains formed by amino acids 26–47 and
317–332 correspond well with those of other species and
participate in ligand-receptor interaction in the rat [37, 38].
Furthermore, the conserved cytoloop II has been suggested
to be a putative site for interaction with G proteins [39].
Similar to the mouse FSHR, hamster FSHR also contains
11 conserved cysteine residues and may participate in the
disulfide binding between exoloops I and II in other G pro-
teins [12]. Several serine, threonine, and tyrosine phos-
phorylation sites also correspond to those of other species
[12]. All these features allow us to contend that the nucle-
otide and deduced amino acid sequences truly represent
FSHR, and that the hamster FSHR mRNA is derived from
1586 ZHANG AND ROY
FIG. 7. Time course of FSH effect on the relative levels of FSHR mRNA
in the hamster ovary. Saline or FSH was injected at 0800 h on Day 4,
and ovaries were collected at indicated times. For the 12-h group, phe-
nobarbitone was injected s.c. at 1300 h to block the endogenous gonad-
otropin surge. Bars with same letter are significantly (P Ͻ 0.05) different
from each other.
10 exons, similar to that reported for other species, includ-
ing human [33].
Because the 5Ј-RACE strategy does not allow truncated
mRNA to be amplified but does allow the 5Ј-oligo primer
to be ligated at the 5Ј-cap site, the putative transcription
start site of the hamster FSHR mRNA likely is located close
to nucleotide Ϫ109, and the 5Ј-UTR of the hamster FSHR
mRNA is 109-bases long. Whereas sequencing of the 5Ј-
flanking region of the mouse FSHR exon 1 from a genomic
library reveals a 500-base 5Ј-UTR [40], genomic cloning
of the human FSHR exon 1 containing the 5Ј-flanking re-
gion has demonstrated five major transcription start sites,
of which the major one is located close to nucleotide Ϫ99
[41]. Genomic cloning of the 5Ј-flanking region of the ham-
ster FSHR will be needed to predict definitive transcription
start site(s); however, the hamster FSHR cDNA sequence
from Ϫ1 to Ϫ109 bases matches well with the correspond-
ing sequences of the mouse and rat.
Analysis of the exon-intron structure of the FSHR gene
indicates the possibility for the formation of alternately
spliced transcripts. In fact, several alternately spliced FSHR
transcripts have been identified in all mammals [17, 33, 42,
43], including the hamster (unpublished observation), by
reverse transcription-PCR amplification of gonadal RNA.
However, in the present study, Northern blot analysis re-
veals only one transcript of approximately 2.5 kb, which
matches well with the 2392-bp FSHR cDNA. Because the
primary transcript of FSHR has a relatively low abundance
in all species, the failure to detect alternately spliced FSHR
transcripts in hamster ovarian RNA likely is caused by low
sensitivity of the Northern hybridization technique. Identi-
fication of a single FSHR transcript in rat ovarian RNA by
Northern hybridization has also been reported [16]. It is,
however, important to remember that only single functional
FSHRs, corresponding to the full-length cDNA, have so far
been identified in the gonads of all species studied [17, 33].
Therefore, identification of only the full-length FSHR
cDNA in the hamster ovary by Northern hybridization ful-
fills the objective of the present study. The size of the ham-
ster FSHR cDNA correlates well with those of the rat, pig,
human, and rabbit [33].
The increases in the levels of FSHR mRNA with pro-
gress of the estrous cycle correlate well with the develop-
ment of antral follicles in the hamster ovary. In the hamster,
atretic antral follicles remain in the ovary on Day 1, and a
new cohort of antral follicles appears on Day 2 and grows
continuously to become the graafian follicles by the morn-
ing of Day 4 (proestrus) [1]. Analysis of serum levels of
FSH in the cyclic hamsters reveals a sharp fall in the hor-
mone levels by the second half of Day 1 to reach consis-
tently low levels by Day 2 through the morning of Day 4
[23], which correspond to follicular growth with a concur-
rent increase in FSHR in the granulosa cells [44]. Dosage
of eCG, which induces in vivo antral follicular development
in the rat, also upregulates FSHR transcripts in the ovary
[15, 15, 22, 45]. Besides, low doses of FSH increase the
levels of FSHR mRNA in cultured rat [46, 47] and porcine
[19] granulosa cells. In hypophysectomized, estrogen-treated
rats, FSH administration results in increased levels of
FSHR mRNA and FSH binding [20]. All these lines of
evidence suggest that low levels of FSH may favor in-
creased levels of FSHR mRNA during follicular growth,
with a concurrent increase in FSHR in the granulosa cells.
The decline in ovarian FSHR mRNA levels following
the gonadotropin surge suggest that in contrast to low lev-
els, high levels of FSH suppress ovarian mRNA levels.
Moreover, LH may also influence the decline, because its
levels also increase during the gonadotropin surge [1]. Sig-
nificant reduction in levels of FSHR mRNA following the
gonadotropin surge in the rat has been reported [45]. In-
volvement of the gonadotropin surge in the down-
regulation of FSHR mRNA levels on the evening of pro-
estrus is further evident by the ability of phenobarbitone to
block the decline in ovarian FSHR mRNA levels. Pheno-
barbitone blocks the preovulatory gonadotropin surge in the
hamster [22]. Interestingly, whereas the second FSH surge
has been suggested to be important for follicular recruit-
ment in the hamster and rat [28] and for inhibin production
in the rat [48], it may not be responsible for the lowered
levels of ovarian FSHR mRNA observed on estrus. How-
ever, it is important to realize that very few healthy, large
antral follicles, which are the primary contributors of high
levels of FSHR mRNA in the ovary, exist on estrous morn-
ing. Therefore, future studies will examine the role of the
second FSH surge in regulating ovarian FSHR mRNA lev-
els. Although LH has been shown to suppress FSHR tran-
script levels in the rat [16], FSH alone may play a major
role in this process. This is evident from the ability of high-
ly pure ovine FSH as well as recombinant human FSH to
mimic the effect of the gonadotropin surge. Similar results
have also been reported for rat FSHR mRNA [17]. Fur-
thermore, recombinant FSH is capable of inducing ovula-
tion in hypophysectomized rats [20]. Collectively, all these
lines of evidence suggest that FSH, by a biphasic modality,
regulates FSHR mRNA levels in hamster ovarian follicles.
Whereas low doses may induce transcription of the FSHR
gene, mRNA stability, or both, high doses may stimulate
the rate of mRNA degradation as well. These biphasic ac-
tions of FSH, in turn, may be regulated by cyclic AMP
along with other intraovarian paracrine factors [17, 33].
In summary, we have identified a complete cDNA se-
quence of the hamster FSHR and obtained the deduced
amino acid sequence, which show all the characteristics of
a complete FSHR molecule. We have also shown changes
in the levels of ovarian FSHR mRNA during the hamster
1587 EFFECT OF GONADOTROPINS ON FSHR mRNA EXPRESSION
estrous cycle and established a role for FSH in modulating
the levels of its own receptor mRNA in the ovary. These
results will help in future studies aimed at understanding
the mechanisms and intraovarian factors that are involved
in regulation of FSHR mRNA levels in the hamster.
ACKNOWLEDGMENT
We thank Dr. A.F. Parlow (National Pituitary Program) for generously
providing the ovine FSH at a subsidized price.
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