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BOR - Papers in Press, published online ahead of print January 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026898
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BIOLOGY OF REPRODUCTION 70, 1580–1588 (2004)
DOI: 10.1095/biolreprod.103.026898
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Downregulation of Follicle-Stimulating Hormone (FSH)-Receptor Messenger RNA Levels in the Hamster Ovary: Effect of the Endogenous and Exogenous FSH1

Yi-Ming Zhang3, and Shyamal K. Roy2,3,4

Departments of Physiology and Biophysics3 Obstetrics and Gynecology,4 University of Nebraska Medical Center, Omaha, Nebraska 68198-4515


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 hamster 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 delineate 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 strategy 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 levels of FSHR mRNA were assessed by Northern blot hybridization. Cloned FSHR cDNA consists of a reading frame corresponding 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 transmembrane 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 attenuation 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 receptor, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicular development depends on FSH action [1]. Lack of FSH function because of a null mutation in the FSHß [2], or FSH-receptor (FSHR) [3] gene, or hypophysectomy [1] in rodents results in impaired follicular development, leading to ovulation failure and infertility. Similarly, neutralization of serum FSH by an anti-FSH serum results in cessation of primordial follicle formation in 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 extracellular 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 transmembrane and the C-terminal cytoplasmic domain is encoded 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 information has led to a significant understanding of the pathophysiology of FSHR gene expression [5, 10–12]. Because FSH has significant roles in hamster ovarian folliculogenesis [1, 4, 13, 14], identifying the nucleic acid and amino acid sequences of hamster FSHR is essential to understanding the regulation of FSHR gene expression in hamster gonads.

Whereas high doses of FSH have been shown to downregulate 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 recombinant human FSH leads to suppression of FSHR mRNA levels in the granulosa cells [20]. Because granulosa cells differentiate into LH-responsive, progesterone-producing 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 information, it is unclear whether the in vivo FSH surge influences the levels of FSHR mRNA in the hamster ovary during 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 exogenous, influences ovarian FSHR mRNA levels. This information will be particularly relevant to understanding how preantral follicles grow after the preovulatory gonadotropin surge [24, 25]. The objectives of the present study were to identify the complete cDNA sequence of the hamster FSHR, to determine the steady-state levels of FSHR mRNA in the ovary during the estrous cycle, and to evaluate the effects of endogenous and exogenous FSH on FSHR mRNA levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Adult golden female hamsters were purchased from Sasco (Madison, WI) and maintained in a climate-controlled room with a 14L:10D photoperiod 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 5'- and 3'-rapid amplification of cDNA ends (RACE) kit and cDNA cloning kit (PCRII TOPO) were from Invitrogen (Carlsbad, CA). All other chemicals were either from Sigma Chemical Company (St. Louis, MO) or Fisher Scientific.

5'- And 3'-RACE Cloning of Full-Length HamsterFSHR 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 corresponding to any putative DNA contamination was evident.

Two FSHR cDNAs, one containing a 3'-untranslated region (UTR) plus a part of the poly A tail (henceforth called 3'-FSHR) and the other containing a complete 5'-UTR (henceforth called 5'-FSHR), were generated using GeneRacer Kit (Invitrogen) according to the manufacturer's instructions. Briefly, to synthesize a single-stranded 3'-FSHR cDNA, 5 µg of total ovarian RNA were denatured at 65°C for 5 min and then mixed with 2.5 µM 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 µl and incubated at 60°C for 60 min, followed by 5 min at 85°C. The RNA template was removed from the mixture by adding 2 U of RNase H, followed by a 20-min incubation at 37°C. Double-stranded 3'-hamster FSHR cDNA was synthesized by polymerase chain reaction (PCR) in a 50-µl reaction mixture containing 1 µl of reverse transcription product, 0.4 µM GeneRacer 3'-primer (complementary to the last 25 bases of the GeneRacer oligo dT), 0.2 µg of a forward primer (F) (Table 1), 2.75 mM MgCl2, 0.2 mM dNTPs, and 2.5 U of Taq polymerase. After a 4-min denaturation step at 94°C, PCR was continued for 30 cycles, followed by a final extension at 72°C for 10 min. The PCR conditions were 1.5 min at 94°C, 1 min at 58°C, and 2.5 min at 72°C. To synthesize a single-stranded 5'-FSHR cDNA, a 5'-oligo (GeneRacer RNA oligo [dT]) (Table 1) was ligated to the 5'-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 5'-hamster FSHR cDNA was synthesized by PCR using R2 and GeneRacer 5'-primers (Table 1). The PCR conditions were 4 min denaturation at 95°C followed by 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C followed by a 10 min extension at 72°C. 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.


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  		TABLE 1. Various primers used to clone hamster FSHR cDNA

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 3'-FSHR) or pCR4-TOPO (for 5'- FSHR) vector (Invitrogen) and transformed in TOPO10 cells (Invitrogen). White colonies were selected under ampicillin, and after thorough screening, 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 sequences of 3'- and 5'-FSHR cDNA fragments were determined by automated DNA sequencing (Genomics Sequencing Service; Qiagen). The sequences were overlapped to derive the full-length hamster FSHR cDNA, including the 5'- and 3'-UTRs. Analyses of nucleotide sequence and predicted amino acid composition of hamster FSHR were carried out using the Vector NTI (Invitrogen) and GCG (Accelrys, San Diego, CA) programs. 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 hybridization, a 504-bp cDNA template, corresponding to 477–981 bases, was generated by PCR from the 3'-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 (Promega, Madison, WI).

Ovarian FSHR mRNA Levels Throughoutthe 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 N2, and stored at –80°C 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 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 µg of denatured total RNA were fractionated in 1% agarose-formaldehyde gels, capillary transferred to Nytran membrane, ultraviolet 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 2x 107 cpm [32P]CTP-labeled FSHR cRNA probe, 40% formamide, 0.5x Denhardt reagent, 7% SDS, and 10% dextran at 60°C in a Hybaid hybridization 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. Finally, 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 5'- and 3'-UTRs for future examination of FSHR mRNA expression in the granulosa cells. The 5'-RACE strategy allowed selection of only those mRNA molecules that had an intact 5'-cap structure and eliminated all truncated mRNA and non-mRNA species, thus assuring the identification of a complete 5'-UTR, which started from the most probable transcription start site. Similarly, binding of the oligo dT primer at the 3'-end of the FSHR mRNA assured the identification of a complete 3'-UTR. The RACE strategy followed by sequencing revealed a 2392-bp cDNA corresponding to FSHR mRNA, which contained a 109-bp 5'-UTR upstream of the translation start site, a 2082-bp open reading frame, a stop codon, and a 198-bp 3'-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 5'-UTR of the hamster FSHR cDNA had 85% and 80% sequence similarities with the corresponding 5'-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 signal peptide, seven transmembrane domains, four conserved putative sites for N-linked glycosylation, 11 cysteine residues, and other putative posttranslational modification sites (Fig. 2 and Table 2).



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  		FIG. 1. Nucleotide sequence of the hamster FSHR cDNA showing 5'- and 3'-untranslated regions and a single open reading frame identified by 5'- and 3'-RACE strategy. Sequence corresponding to signal peptide and stop codon are in bold. Beginning 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 nucleotide –109. +1, Putative translation initiation site.



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  		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


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  		TABLE 2. Putative posttranslational modification sites

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, adrenal gland, or liver, indicating the specificity of the signal (Fig. 3). However, no alternately spliced FSHR transcript could be detected in the ovarian sample.



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  		FIG. 3. Northern hybridization analysis of hamster FSHR mRNA. A) Phosphorimage showing a [32P]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

Ovarian FSHR mRNA Levels Throughoutthe 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, ovarian 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).



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  		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 < 0.05) different from each other

Effect of Endogenous FSH Surge on Ovarian FSHRmRNA 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 phenobarbitone 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).



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  		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 < 0.05) different from each other

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 rationale 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 present 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. 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, without the presence of LH, can downregulate FSHR mRNA levels in the hamster ovary.



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  		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 < 0.05) different from each other. oFSH, ovine FSH; rhFSH, recombinant human FSH

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. Significant 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 effect of the endogenous increase in FSH levels, which maintained the decline up to the morning of Day 1.



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  		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, phenobarbitone was injected s.c. at 1300 h to block the endogenous gonadotropin surge. Bars with same letter are significantly (P < 0.05) different from each other


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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. Because FSH plays an important regulatory role in hamster preantral folliculogenesis, regulation of FSHR mRNA levels 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 amino 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 amino 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 species. 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]. Furthermore, three conserved putative sites for N-linked glycosylation and 11 cysteine residues in the extracellular domain 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- 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 reported for human and monkey FSHR [17]. Therefore, hamster 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 proteins [12]. Several serine, threonine, and tyrosine phosphorylation sites also correspond to those of other species [12]. All these features allow us to contend that the nucleotide and deduced amino acid sequences truly represent FSHR, and that the hamster FSHR mRNA is derived from 10 exons, similar to that reported for other species, including human [33].

Because the 5'-RACE strategy does not allow truncated mRNA to be amplified but does allow the 5'-oligo primer to be ligated at the 5'-cap site, the putative transcription start site of the hamster FSHR mRNA likely is located close to nucleotide –109, and the 5'-UTR of the hamster FSHR mRNA is 109-bases long. Whereas sequencing of the 5'- flanking region of the mouse FSHR exon 1 from a genomic library reveals a 500-base 5'-UTR [40], genomic cloning of the human FSHR exon 1 containing the 5'-flanking region has demonstrated five major transcription start sites, of which the major one is located close to nucleotide –99 [41]. Genomic cloning of the 5'-flanking region of the hamster FSHR will be needed to predict definitive transcription start site(s); however, the hamster FSHR cDNA sequence from –1 to –109 bases matches well with the corresponding 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 reveals 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. Identification 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 fulfills the objective of the present study. The size of the hamster FSHR cDNA correlates well with those of the rat, pig, human, and rabbit [33].

The increases in the levels of FSHR mRNA with progress of the estrous cycle correlate well with the development 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 morning of Day 4 (proestrus) [1]. Analysis of serum levels of FSH in the cyclic hamsters reveals a sharp fall in the hormone levels by the second half of Day 1 to reach consistently low levels by Day 2 through the morning of Day 4 [23], which correspond to follicular growth with a concurrent 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 increased 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 levels, 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]. Significant reduction in levels of FSHR mRNA following the gonadotropin surge in the rat has been reported [45]. Involvement of the gonadotropin surge in the down-regulation of FSHR mRNA levels on the evening of proestrus is further evident by the ability of phenobarbitone to block the decline in ovarian FSHR mRNA levels. Phenobarbitone blocks the preovulatory gonadotropin surge in the hamster [22]. Interestingly, whereas the second FSH surge has been suggested to be important for follicular recruitment 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. However, 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 morning. Therefore, future studies will examine the role of the second FSH surge in regulating ovarian FSHR mRNA levels. Although LH has been shown to suppress FSHR transcript levels in the rat [16], FSH alone may play a major role in this process. This is evident from the ability of highly 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]. Furthermore, recombinant FSH is capable of inducing ovulation 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 actions 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 sequence 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 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.


   ACKNOWLEDGMENTS
 
We thank Dr. A.F. Parlow (National Pituitary Program) for generously providing the ovine FSH at a subsidized price.


   FOOTNOTES
 
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 deposited in the GenBank (accession no. AY509907). Back

2 Correspondence: Shyamal K. Roy, Departments of OB/GYN and Physiology and Biophysics, DRC 5013, University of Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu Back

Received: 22 December 2003.

First decision: 20 January 2004.

Accepted: 22 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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