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Regular Article |
a Leland J. and Dorothy H. Olson Center for Women's Health, Departments of Ob/Gyn and Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
ABSTRACT
The hormonal regulation of ovarian transforming growth factor-ß (TGF-ß) type I receptor (TßRI) and TßRII messenger (mRNA) expression was evaluated using cyclic and hypophysectomized hamsters. Northern blot analysis revealed that three TßRI and one TßRII gene transcripts were expressed in the hamster ovary. Reverse transcription-polymerase chain reaction quantitation revealed that receptor mRNA was differentially expressed during the estrous cycle. Although, mRNA levels for both receptor types increased steadily up to Day 4:0900 h, a sharp decline occurred following the gonadotropin surge. In fact, receptor mRNA started declining by Day 4:1200 h, long before the gonadotropin surge; however, only TßRI mRNA levels recovered partially by 1500 h to fall again by 1600 h. Although hypophysectomy preferentially reduced TßRII mRNA levels, gonadotropins as well as ovarian steroids significantly induced TßRI and TßRII mRNA expression within 48 h and 24 h, respectively; 5
-dihydrotesterone (DHT) induced only TßRII mRNA. The induction of ovarian TßRI and TßRII mRNA by estradiol-17ß or progesterone was severely attenuated by dexamethasone. A marked increase in serum cortisol coincided with the periovulatory rise in serum gonadotropins. These results suggest that the increase in TGF-ß receptor mRNA expression correlates with gonadotropin-induced ovarian follicular development during the estrous cycle. Moreover, receptor mRNA expression is critically and differentially regulated by gonadotropins as well as ovarian steroids. Most importantly, glucocorticoid appears to play a critical modulatory role in the temporal expression of receptor mRNA in the ovary, hence, controlling folliculogenesis.
Recently, we have shown that transforming growth factor-ß (TGF-ß) receptor type II (TßRII) protein is expressed in hamster follicular as well as in interstitial cells, and FSH, estrogen, and 5-dihydrotestosterone (DHT), a nonaromatizable androgen, up-regulate receptor protein expression [1]. The expression of TGF-ß receptor protein and mRNA have also been detected in cultured porcine granulosa cells [2]. Numerous studies have documented that ovarian cells of a variety of species express TGF-ß gene transcripts and their ligands [3–10], and exogenous TGF-ß significantly influences the functions of both granulosa and thecal cells [10–12]. All these lines of evidence clearly establish the presence of a TGF-ß system in the mammalian ovary and a significant role of TGF-ß in ovarian follicular development.
Transforming growth factor-ß belongs to a multigene family and, depending on the cell types, influences cell growth, differentiation, motility, organization, and death [13, 14]. The physiological action of TGF-ß is mediated by a ligand-induced cell surface receptor complex consisting of TGF-ß receptor type I and type II [14, 15]. Both type I and II receptor proteins contain an amino-terminal signal sequence, a cysteine-rich extracellular ligand binding domain with N-linked glycosylation sites, a single hydrophobic transmembrane domain, and a cytoplasmic kinase domain [13]. The expression of TßRII mRNA in the rat ovary [16], and TßR-I and -II mRNA in cultured porcine granulosa cells [2] has been documented. Hamster follicular development is significantly affected by TGF-ß [16, 17] and TßRII protein expression in the hamster ovary is critically influenced by gonadotropins and sex steroids [8]. However, it is unclear whether gonadotropins and steroid hormones also modulate ovarian TGF-ß receptor mRNA levels. Species-specific receptor cDNAs are critical for the quantitation of mRNA levels; however, no information is available for the nucleic acid sequence of hamster TGF-ß receptor cDNA. Therefore, cDNAs corresponding to the kinase domain of hamster TGF-ß receptor type I and type II receptors have been generated and used in the present study. The objective of the present study was to study the role of hormonal interaction on ovarian TßR mRNA expression during the estrous cycle. The study was done using the whole ovary rather than any single compartment because the first goal was to identify whether hormones influence the overall expression of ovarian receptor mRNA, which reflects the cumulated interaction of different cell types.
MATERIALS AND METHODS
Golden hamsters (90–100 g) were purchased from SASCO (Madison, WI) and maintained in a 14L:10D cycle in a climate-controlled facility according to IACUC and USDA guidelines. All oligodeoxynucleotide primers were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Reverse transcription-polymerase chain reaction (RT-PCR) chemicals were obtained from Life Technologies (Rockville, MD), Boehringer/Roche Molecular Biochemicals (Mannheim, Germany) or Amersham/Pharmacia (Piscataway, NJ), and Zeta Probe nucleic acid transfer membrane was from Bio-Rad (Hercules, CA). TriReagent for RNA extraction was from MRC, Inc. (Cincinnati, OH), plasmid vector pGemT-easy and riboprobe kit were from Promega (Madison, WI), [32P]-CTP (specific activity 1000 Ci/mmol) and [32P]-
-ATP (specific activity 7000 Ci/mmol) were from ICN Radiochemicals (Costa Mesa, CA). Cortisol assay kit was from Diagnostic Products Corporation (Los Angeles, CA). All other molecular grade chemicals were obtained from Life Technologies. Gonadotropins were generously provided by the National Hormone Program (NIH) and Dr. A.F. Parlow (Stanford, CA); progesterone antibody was generously provided by Dr. D.C. Johnson (University of Kansas Medical Center, Kansas City, KS); and estradiol-17ß (E2) antibody was a gift from Dr. M. Kumar (Kansas State University, Manhattan, KS).
Experiment 1: Development of Hamster TßRI and TßRII cDNA by RT-PCR
Hamster ovarian poly(A)+ RNA was used for generating cDNA corresponding to the kinase domain of TßRI and TßRII. The forward (5'-GGTCTTGCCCATCTTCACATG-3') and reverse (5'-GTCGAGCAATTTCCCAG-3') primers for TßRI receptor cDNA were selected from the homologous region of mouse TßRI [18] and human ALK-5 [19] cDNA. The forward (5'-AAGGCCAAGCTGAAGCAGAAC-3') and reverse (5'-AGCTCTTGAGGTCCCTGTG-3') primers for TßRII receptor cDNA were selected from the homologous region of mouse [20] and human [21] TßRII cDNA. The specificity of the PCR products was verified using restriction digestion and Southern blotting using 5'-[32P]-oligodeoxynucleotides specific for mouse TßRI and TßRII cDNA, respectively. Hamster TßRI and TßRII cDNA were cloned in PGEM-T-easy vector (Promega) and sequenced in an automated DNA sequencer (ABI, UNMC Genetic Sequence Core). The cDNA sequences of both TßRI and TßRII were compared with that of human, mouse and rat for similarity and with other cDNA sequences in the GeneBank to ensure specificity using GCG program (UNMC genetic analysis core). The TßRI and TßRII cDNAs were transcribed using [32P]-CTP and a riboprobe kit (Promega), and the probes were used for Northern hybridization evaluation of hamster ovarian TßR mRNA. The forward and reverse primers were used subsequently for semiquantitative assessment of the levels of receptor mRNA in the ovary.
Next, hamster ovarian poly(A)+ RNA was extracted using TriReagent and oligo(dT)+ cellulose (Collaborative Research, Bedford, MA) according to the manufacturers' instructions and used for Northern analysis of TßRI and TßRII as described previously [22] with some modifications. Briefly, 10 µg of poly(A)+ RNA was fractionated in a denaturing formaldehyde agarose gel, transferred to a Zeta-probe membrane and hybridized overnight at 68°C in 40% formamide/6x standard saline citrate in the presence of either [32P]-sense or antisense cRNA, and the signal was determined after exposing the membrane to a NEN-DuPont Reflection film for 48 h.
Experiment 2: Analysis of Ovarian TßRI and TßRII mRNA During the Estrous Cycle
Animals were checked for at least three consecutive estrous cycles, and ovaries were collected at 0900 h on each day of the estrous cycle (Day 1 = estrous; Day 4 = proestrous) and at 1600 h on Day 4 to delineate TßR and S4 (hamster ribosomal protein) mRNA levels under endogenous hormonal milieu and after the periovulatory gonadotropin surge. S4 was selected for normalization and verification of the specificity of TGF-ß receptor mRNA expression.
Because ovarian steroid hormone profiles change dramatically during the periovulatory period on Day 4, in a separate experiment, ovaries from proestrus hamsters were collected hourly from 1200 h (before the LH surge) through 1700 h (after the LH surge) and the RNA was evaluated for TßRI and TßRII mRNA levels. The levels of estradiol-17ß (E2) and progesterone (P) were determined by hormone-specific RIA as described before [1, 23].
Experiment 3: Effect of Exogenous Gonadotropinsand Steroid Hormones on Ovarian TßRI and TßRIImRNA Expression
Animals were hypophysectomized (HX) at Day 1:0900 h under Nembutal (sodium pentobarbital) anesthesia as described before [1, 24]. Ten days after the surgery, hamsters were injected subcutaneously (s.c.) with either vehicle (0.5 mg BSA in saline), 10 µg ovine-FSH (NIH-FSH-19) twice daily for 2 days or 10 U hCG (NIH-CR-125) with or without FSH, and killed 48 h later. Human chorionic gonadotropin was used because of its longer half life than LH. A separate group of HX animals received a single s.c. injection of either 0.5 mg of E2, 1 mg of progesterone (P), 0.5 mg DHT, or 0.5 mg E2 followed 6 h later by 1 mg of progesterone. Steroid hormone-injected animals were killed 24 h after the first injection and ovaries were retrieved to determine the levels of receptor mRNA. Although the dosage of steroids appeared to be pharmacological, circulating levels of E2 and P were close to the levels found on the morning of Day 4 and Day 1, respectively. The goal was to evaluate any effect of steroid hormones on ovarian TßR mRNA levels. We used DHT to determine the direct effect of androgen on ovarian TßR mRNA levels and because DHT significantly influences TßRII protein expression in the hamster ovary [1]. Besides, we have used these dosages of steroid hormones to evaluate TßRII protein expression in the hamster ovary [1].
Serum levels of progesterone and E2 during the periovulatory period of cyclic hamsters, and of HX-hamster treated with or without steroid hormones were measured using steroid specific RIA as described previously [1, 23].
Experiment 4: Changes in the Receptor mRNA During the Periovulatory Period
In order to identify a window when critical changes in receptor mRNA levels occur during the periovulatory period, ovaries were collected from proestrous hamsters at 0900 h and at hourly intervals from 1200 h through 1700 h. The levels of receptor mRNA expression were evaluated.
Experiment 5: Interaction of Glucocorticoid and Ovarian Steroids Regulating Ovarian TßR mRNA Expression
To determine a possible mechanism of endogenous and exogenous gonadotropin regulation of ovarian receptor mRNA expression, 10-day HX hamsters were injected with a single s.c. injection of either 1 mg dexamethasone (DEX), 1 mg P, 0.5 mg E2, a combination of P and DEX, E2 and DEX, or P, E2, and DEX. Ovaries were retrieved 24 h later for receptor and S4 mRNA analysis. To correlate the effect of exogenous glucocorticoid with in vivo situation, serum levels of free cortisol were detected throughout the estrous cycle and during the periovulatory surge using a coat-a-count kit that used [125I]-cortisol as a tracer and required no solvent extraction of the serum. The assay had a sensitivity of 0.25 ng cortisol per tube and had the following cross-reactivity with other steroids: aldosterone 0.03%, corticosterone 0.94%, estrone 0.01%, pregnenolone 0.02%, progesterone 0.02% and tetrahydrocortisol 0.34%. All serum samples were assayed in a single run to avoid interassay variation. The intraassay variation was approximately 5%. Serum from long-term HX hamsters was included in the assay as an internal control.
RT-PCR Amplification of Hamster Ovarian TßR and S4 mRNA
Semiquantitative RT-PCR was done following a basic RT-PCR protocol described by Das et al. [25] that was modified for our study. Briefly, 0.5 µg of total RNA was reverse transcribed for 40 min at 38°C in the presence of 1.5 µM of random hexamers (Amersham/Pharmacia, Arlington Heights, IL) and 100 U of MMLV-RT in a final volume of 7 µl. Because the data were expressed as a ratio of receptor and S4 mRNA to evaluate the specificity of receptor mRNA expression, a 1.5-µl aliquot of the RT product was used each for TßRI, TßRII and S4 cDNA amplification to avoid the variation in RNA amount. The PCR mixture contained 0.7 µM gene specific forward and reverse primers, dNTPs, BSA, and 1 U of Taq DNA polymerase (Promega). The amplification was continued for 30 cycles and a typical cycle consisted of 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C. The PCR was optimized by verifying the linearity of cDNA synthesis for a total of 60 cycles. The synthesis of cDNA was linear from 20 through 60 cycles for receptor as well as S4 (Fig. 1); hence, a 30-cycle run was selected to stay within the linear range of amplification. The fidelity of the PCR was verified using controls 1) containing no RT reaction product, and 2) RT reaction product formed in the presence of RNase A–treated RNA. The purity of the RNA was verified using RT reaction product that was devoid of MMLV-reverse transcriptase. No PCR product was generated in any of the control tubes ensuring the purity of the RNA and cDNA products, and the fidelity of the RT-PCR.
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The forward (5'-GGCGATGAAGTCAAGAAG-3') and reverse (5'-CCCAAGTTAGCACCTCC-3') primers for hamster ribosomal protein, S4, were selected from the published cDNA sequence [26].
Southern Blotting and Semiquantitative Analysis of Ovarian TßR and S4 cDNA by Phosphorimaging
Southern hybridization was done according to Schatz et al. [27]. In brief, PCR product was fractionated in a 1% agarose-ethidium bromide gel under 1x TBS, pH 8.0 (Tris-HCl, pH 8.0, boric acid, and EDTA), visualized by UV-transillumination, denatured by alkaline hydrolysis, and neutralized, capillary transferred to a Zeta probe membrane, UV-cross linked, and hybridized overnight at 42°C with a [32P]-end labeled oligodeoxynucleotide probe corresponding to respective cDNA. The probes were labeled using a Maxiscript kit (Ambion, Austin, TX). The membrane hybridized with labeled S4 probe was exposed to the phosphor screen for 5 min while those hybridized with labeled receptor probe were exposed for 20 min. This was necessary because of the higher concentration of S4 mRNA in the total RNA preparation. The signal was quantified using a Packard cyclone phosphorimager. After background subtraction, total DLU (digital light units) for the receptor as well as S4 mRNA was reduced to DLU/min, and the results were expressed as the DLU ratio of the receptor mRNA versus S4 mRNA. This conversion was possible because of the higher signal absorption capacity of the phosphor screen.
Data Analysis
Ovarian RNA from four different hamsters was analyzed to get a total number of observation (n) = 4. Values for each group were analyzed by two-way ANOVA and Fischer's exact test using Statview statistical software (Abacus Concepts, Inc.). The level of significance was at 5%.
RESULTS
Amplification of Hamster TßRI and TßRII cDNA
The primer sets for TßRI (lanes 1–3) and TßRII (lanes 5–7) generated a predicted 306-bp and a 372-bp cDNA, respectively, while those for S4 (lanes 9–11) generated a predicted 398-bp cDNA (Fig. 2). No cDNA was detected when reverse transcriptase was omitted from the RT reaction, verifying the purity of the RNA (Fig. 2, lanes 4 and 8). Likewise, no cDNA was detected without RNA or with RNase-treated RNA (data not shown). The difference in the levels of TßRI and TßRII mRNA in the hamster ovary was apparent. Hamster TßRII cDNA sequence was 88% similar to corresponding human [21] and rat [28] TßRII sequences, and hamster TßRI cDNA sequence was 92% similar to corresponding human [19] and mouse [18] TßRI cDNA sequences (data not shown). Northern analysis revealed the presence of one 4.37-kb TßRII (Fig. 3) and three (a major 7.8-kb and two minor, 4.37- and 2.28-kb) TßRI gene transcripts in the hamster ovary (Fig. 3). Semiquantitative RT-PCR evaluation revealed that TGF-ß receptor mRNA were differentially expressed in the hamster ovary. For each day of the estrous cycle, ovarian TßRII mRNA levels at 0900 h were at least threefold higher than that of TßRI mRNA (Fig. 4). The relative amount of TßRII mRNA increased steadily from Day 1:0900 h to reach a significantly (P < 0.05) high level at Day 4:0900 h. In contrast, a significant (P < 0.05) increase in TßRI mRNA levels occurred at Day 3:0900 h. Receptor mRNA levels declined significantly (P < 0.05) following the periovulatory gonadotropin surge (Fig. 4).
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On proestrous, serum levels of E2 were high compared to P at 1200 h (Fig. 5). Progesterone levels did not change until 1400 h, coinciding with the LH surge [29], and increased steadily through 1700 h. Although serum E2 levels also peaked by 1600 h, the levels started declining by 1700 h resulting in a P-dominated environment (Fig. 5). Coinciding with changes in serum steroid hormone levels, TßRI mRNA levels increased gradually until 1500 h before declining further by 1600 h. In contrast, TßRII mRNA levels decreased steadily from 1200 h through 1600 h to reach a plateau by 1700 h (Fig. 6B). Hypophysectomy on Day 1:0900 h resulted 10 days later in a significant reduction in ovarian TßRII but not TßRI mRNA levels compared to that of cyclic Day 1:0900 h (Fig. 7). Follicle-stimulating hormone or hCG significantly increased the steady-state mRNA levels for both types of receptor (Fig. 7). Although, hCG-induced TßRII mRNA expression was significantly more than that of FSH, TßRII mRNA levels fell significantly when FSH was administered along with hCG (Fig. 7). While E2 increased TßRI mRNA expression, the effect was significantly reduced in the presence of P (Fig. 7). In contrast, a synergistic effect of E2 and P was noted for TßRII mRNA levels (Fig. 7). On the other hand, DHT significantly increased only TßRII mRNA levels (Fig. 7). Despite the known induction of preantral and antral follicle development in the hamster ovary by FSH [1], only hCG slightly increased serum P levels (Fig. 8). Follicle-stimulating hormone, however, was capable of slightly increasing serum E2 level regardless of the presence of hCG (Fig. 8). Exogenous steroid hormones maintained the serum levels within the nanogram range (Fig. 8) that correlated well with the pattern of serum levels of P at Day 1:0900 h and E2 at Day 4:0900 h. Administration of high dosages of steroids was necessary to maintain steady systemic levels of ovarian steroids. The DHT had no effect on serum levels of P and E2 (Fig. 8).
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It was rather paradoxical that in cyclic hamsters, ovarian TßRI and TßRII mRNA levels decreased following the periovulatory gonadotropin surge but the levels of both mRNA increased significantly in HX hamsters treated with exogenous gonadotropins. To delineate the possible mechanism(s) of such a paradoxical effect we tested the effect of DEX on the expression of TßRI and TßRII mRNA in the hamster ovary. Consistent with our earlier results (Fig. 7), administration of P alone significantly (P < 0.05) elevated both TßRI and TßRII mRNA levels (Fig. 9), but a combined dose of DEX and P sharply reduced P-induced increase in ovarian TßRI mRNA levels by 6.5-fold and TßRII mRNA expression by 2-fold (Fig. 9). Alone, DEX stimulated (2-fold) basal TßRII mRNA expression but reduced basal TßRI mRNA by 2.6-fold (Fig. 9). Dexamethasone also inhibited E2- and E2-and P-induced TßRI mRNA expression by 10-fold (Fig. 9) and TßRII mRNA levels by 11-fold (Fig. 9). Serum levels of cortisol were modest on Day 1:0900 h and declined further by Day 4:0900 h; however, serum cortisol levels started increasing from Day 4:1200 h onward to reach a peak (P < 0.05) by Day 4:1600 h (Fig. 10), coinciding with the preovulatory gonadotropin surge. Serum cortisol levels declined by 1700 h, albeit the level was still significantly (P < 0.05) higher than that observed on Day 1 through Day 4:0900 h (Fig. 10).
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DISCUSSION
This is the first report of a systematic analysis of hormonal regulation of TGF-ß receptor mRNA expression in the mammalian ovary. The functions of TßRI and TßRII have been widely studied in various cell types [13, 30–36] but virtually no information is available about the hormonal regulation of TGF-ß receptor mRNA expression in the mammalian ovary. Because both TßRI and TßRII are essential for transducing the biological action of TGF-ß [34, 35], analysis of changes in the expression pattern of both of the receptor mRNA in the ovary during the reproductive cycle is critical to understand the hormonal modulation of ovarian functions. Whereas the presence of three TßRI and one TßRII gene transcripts has been documented for porcine granulosa cells [2], the size of hamster TßRII mRNA was closer to that reported for porcine granulosa cell (4.6 kb) [2] and human hepatoma cell line [37, 38]. The significance of the minor transcripts remains unclear.
The results of the present study reflect cumulative changes in receptor mRNA levels for the entire ovary resultant of the activities of several different cell types. However, the information is the first of a kind to establish that reproductive hormones modulate ovarian TGF-ß receptor mRNA levels. The increase in both TßRI and TßRII mRNA levels during the estrous cycle corroborates the findings on TßRII protein expression in the hamster ovary [1]. The preferential rise in TßRII mRNA levels at Day 1:0900 h may be due to the effect of secondary FSH surge that occurs at 2200 h on proestrous [28] and suggests that TßRII and TßRI mRNA expression are differentially influenced by gonadotropins, ovarian steroids, or both.
In the hamster, serum P levels are high on Days 1 and 2 of the estrous cycle while estrogen becomes dominant by Day 4:0900 h [1, 9]. In the present study, temporal changes in serum P and E2 levels during the estrous cycle correlate well with the increased levels of mRNA for both types of TßR. Although a clear-cut differential expression of TßRI and TßRII mRNA is evident in the hamster ovary throughout the estrous cycle, the unique periovulatory expression patterns indicate that additional control mechanisms may exist at that time. This is further evident by the sharp decline in the receptor mRNA levels by 1200 h, while serum levels of ovarian steroids, especially P, do not change, and from the results of studies done on HX hamsters. Whereas the modest reversal of TßRI mRNA expression by 1500 h may be due an increase in serum LH and/or ovarian steroids, the sharp decrease by 1600 h coincides with a very high level of serum progesterone. Because hormones require some latency before their effect can be detected, changes in the steady-state levels of receptor mRNA noted at 1700 h may be the result of increasing levels of P from 1400 h and onward.
The exact reason for the steady decline in TßRI and TßRII mRNA levels on proestrous, especially during the preovulatory gonadotropin surge, is not known. Because, gonadotropins as well as ovarian steroids increase receptor mRNA levels in HX hamster ovaries, it is likely that the observed negative regulation may be exerted by some other factors, particularly cortisol. This conjecture is supported by the evidence that DEX significantly inhibits both P- and E2-induced expression of TßRI and TßRII mRNA in the hamster ovary, and serum cortisol levels start increasing from 1200 h onward to reach a peak by 1600 h on proestrous. Activation of the pituitary-adrenal axis in the afternoon of proestrous [39], and a possible role for glucocorticoid in the timing of mating and ovulation [40] in the rat have been demonstrated. Mahesh and Brann [41] have documented that adrenocorticotropic and gonadotropic axes interact to regulate gonadotropin secretion. Moreover, serum levels of ACTH and corticosterone increase prior to the start of the periovulatory LH surge in the rat [39, 42, 43], and a midcycle increase in ACTH and cortisol has been reported in women [44]. Kerdelhue et al. [45] have demonstrated that serum levels of ACTH and cortisol increase significantly following a single injection of E2-benzoate to ovariectomized cynomolgous monkeys. Administration of ACTH to cyclic hamsters results in an increase in the utero-ovarian blood flow and serum P levels [46]. Recently, more direct evidence about the glucocorticoid inhibition of TßR gene transcription has been put forward by Chang et al. [47] who have shown that either cortisol or DEX significantly inhibits the synthesis of transcription factor CBFa1, which is critical for TGF-ß receptor gene transcription, in bone cells. All these lines of evidence suggest that a periovulatory rise in serum cortisol in the hamster may suppress TGF-ß receptor gene transcription to critically modulate the action of TGF-ß on ovarian cells. Moreover, the transcription machinery for TßRI mRNA synthesis may be more susceptible to cortisol inhibition than that of TßRII as evident from the relatively rapid decline in TßRI mRNA during the periovulatory period. Follicle-stimulating hormone stimulates the expression of TGF-ß2 and TßRII proteins in the hamster ovary [1] and in cultured human preantral follicles [48]. Therefore, intervention by cortisol may be important for temporal regulation of TGF-ß action on follicular cells. Because DEX is more potent than hydrocortisone and does not bind to corticosteroid-binding globulin (CBG), it may have exaggerated the cortisol effect to certain extent. However, it is clear that activation of ovarian glucocorticoid receptors during estrogen and progesterone action is inhibitory to TßR mRNA expression in ovarian cells. Because glucocorticoid directly inhibits TßR gene transcription [47], it is likely that at least part of the DEX-induced decline in the steady-state TßR mRNA levels in the hamster ovary is due to an inhibition of TßR mRNA synthesis. However, further studies are needed to determine this possibility. It is unclear why FSH preferentially attenuates the hCG-induced increase in TßRII mRNA in HX hamsters. Because FSH induces growth while LH causes terminal differentiation of follicles [9], attenuation of hCG action will prevent premature luteinization and/or thecal hyperactivity and allow regulated follicular development. Mural granulosa cells of hamster preovulatory follicles (terminally differentiated) express a very high level of TGF-ß ligand [6], TßRII [1] and LH receptor [8]. Therefore, premature overinduction of TßR in granulosa cells of developing follicles will be detrimental to follicle growth. Increased follicular production of E2, P, and androgen is the hallmark of follicle differentiation, and all these steroids significantly induce TßRII mRNA, and TßRII binds TGF-ß ligand [13]. Present results also document a direct stimulatory effect of androgen on ovarian TGF-ß receptor mRNA expression. Dihydrotestosterone has been shown to induce insulin-like growth factor type I and primary follicle development in the rhesus monkey [49].
In summary, the results of the present study provide strong evidence that the expression of TGF-ß receptor mRNA in the hamster ovary is positively regulated by gonadotropins and ovarian steroid hormones; however, the action of these hormones is temporally and critically modulated by cortisol.
ACKNOWLEDGMENTS
I thank the National Pituitary Program and Dr. A.F. Parlow for their generosity in providing the FSH and LH. I also thank Dr. S.K. Das, University of Kansas Medical Center for helping me to set up the RT-PCR technique and Dr. S.K. Dey for allowing me to use his laboratory during the trial.
FOOTNOTES
First decision: 29 December 1999.
1 This study was supported by a grant (HD28165) from NICHHD and the Olson Foundation of Omaha. 
2 Correspondence: S.K. Roy, Department of Ob/Gyn and Physiology and Biophysics, BH4030, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Accepted: January 28, 2000.
Received: November 22, 1999.
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P. Yang and S. K. Roy Epidermal Growth Factor Modulates Transforming Growth Factor Receptor Messenger RNA and Protein Levels in Hamster Preantral Follicles In Vitro Biol Reprod, September 1, 2001; 65(3): 847 - 854. [Abstract] [Full Text] [PDF]
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J. R. Schwartz and S. K. Roy Developmental Expression of Cytochrome P450 Side-Chain Cleavage and Cytochrome P450 17{alpha}-Hydroxylase Messenger Ribonucleic Acid and Protein in the Neonatal Hamster Ovary Biol Reprod, December 1, 2000; 63(6): 1586 - 1593. [Abstract] [Full Text]
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