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Endocrinology Vol. 140, No. 4 1783-1788
Copyright © 1999 by The Endocrine Society

ARTICLES

Thyroid Hormone Effects on Mouse Oocyte Maturation and Granulosa Cell Aromatase Activity1

S. Cecconi, N. Rucci, M. L. Scaldaferri, M. P. Masciulli, G. Rossi, C. Moretti, M. D’Armiento and S. Ulisse

Departments of Experimental Medicine and Science and Biomedical Technologies, University of L’Aquila (S.C., M.P.M., G.R.), 67100 L’Aquila; the Department of Internal Medicine, University of Rome Tor Vergata (M.L.S., C.M.), 00173 Rome; and the Department of Experimental Medicine and Pathology, University of Rome La Sapienza (M.D.), 00161 Rome, Italy

Address all correspondence and requests for reprints to: Dr. Salvatore Ulisse, Department of Experimental Medicine, University of L’Aquila, Via Vetoio, 67100 L’Aquila, Italy. E-mail: ulisse{at}univaq.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study we evaluated the role of T3 on the in vitro processes of mouse cumulus cell-oocyte complex expansion, oocyte meiotic maturation, and granulosa cell aromatase activity. Results obtained from cumuli oophori isolated from immature and adult mice ovaries demonstrated that T3 at all concentrations tested (0.1–100 nM) did not affect basal or FSH-induced cumulus expansion or interfere with oocyte meiotic maturation up to metaphase II stage. On the contrary, T3 inhibited in a time- and dose-dependent manner FSH-induced aromatase activity in cultured granulosa cells obtained from either adult or immature female mice. The half-maximal dose (ED50) of T3 inhibition was 0.87 ± 0.21 nM, which is in agreement with the reported dissociation constant of T3 nuclear receptor (Kd = 0.4–5 nM) in mammalian granulosa cells. Time-course experiments demonstrated higher sensitivity to T3 of adult granulosa cells with respect to immature granulosa cells in culture. Indeed, in immature granulosa cells T3 inhibition became significantly evident only after 6 days of hormonal treatment, whereas in adult granulosa cells the inhibitory effect was present after only 2 days of treatment. (Bu)2cAMP- or 3-isobutyl-1-methyl-xanthine-stimulated aromatase activity was also significantly decreased by T3, thus suggesting that the inhibition was downstream from cAMP formation. Lastly, analysis of aromatase messenger RNA (mRNA) levels by semiquantitative RT-PCR demonstrated the ability of FSH to increase aromatase mRNA level in cultured granulosa cells by 2.4 ± 0.5-fold. In agreement with the effect on enzyme activity, the stimulatory effect of FSH on aromatase mRNA level was greatly reduced after T3 cotreatment. In conclusion, T3 inhibition of aromatase activity may be of physiological relevance in the complex multihormonal regulation of mammalian follicle development and may contribute to explaining the alteration in female reproductive functions after thyroid hormone hypo- or hypersecretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MATURE OOCYTE production and steroid hormone biosynthesis are fundamental processes of the mammalian ovary. These functions are accomplished during follicle development, which represents a complex process under strict hormonal control (1). Pituitary gonadotropins play a major role in the regulation of follicle growth and differentiation (2, 3). In addition, a variety of other hormones and growth factors have been shown to affect ovarian follicle development (4, 5, 6, 7, 8).

In recent years it has become increasingly clear that adequate levels of circulating thyroid hormone (T3) are of primary importance for normal female reproductive functions. Indeed, in both humans and animals, changes in T3 levels result in menstrual disturbances, impaired fertility, and altered pituitary gonadotropin secretion (9, 10, 11, 12, 13). Moreover, T3 has been shown to directly modulate FSH and LH action on steroid biosynthesis in porcine (14, 15) and human granulosa (16, 17) cells in vitro. These findings are further supported by the identification of multiple T3 receptor messenger RNAs (mRNAs) and T3-binding sites in mammalian granulosa and stromal cells (18, 19, 20, 21, 22) and, more recently, in human cumulus cells and oocytes (23).

To better characterize the nature of T3 action in the mammalian ovary, in the present study we have investigated the effects of T3 on the processes of cumulus expansion and oocyte meiotic maturation by using cumulus cell oocyte complexes (COCS) obtained from ovaries of either immature or adult mice. The effects of T3 on basal and FSH-induced aromatase activity in granulosa cells have also been evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM; hyaluronidase; BSA; HEPES; FCS; (Bu)2cAMP, 3-isobutyl-1-methyl-xanthine (MIX); 19-hydroxy-4-androstene-3,17-dione (19OH-androstenedione); insulin, transferrin, and selenium (ITS); 17ß-estradiol (E2); and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). [3H]E2 was purchased from DuPont-New England Nuclear (Boston, MA). Random primer and deoxy-NTP were purchased from Amersham Pharmacia Biotech (Milan, Italy). Moloney murine leukemia virus (M-MLV) reverse transcriptase was purchased from Life Technologies (San Giuliano Milanese, Italy). Taq DNA polymerase was purchased from Promega Corp. (Milan, Italy). The Bradford protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). E2 antiserum was kindly provided by Radim (Pomezia, Italia). Ovine FSH (oFSH) was provided by Dr. A. F. Parlow (NIDDK, Bethesda, MD). T3 was provided by Dr. J. R. Tata (National Institute for Medical Research, London, UK).

COCS collection and in vitro maturation
Immature (22–24 days of age) and adult (60–80 days of age) female mice (CD1, Charles River, Calco (CO), Italy) were used in all experiments. All experimental protocols were approved by the local ethical committee. COCS were collected by puncturing large antral follicles (follicle diameter, >350 µm) of immature or adult mice in DMEM buffered with 20 mM HEPES, pH 7.3, and supplemented with 1 mg/ml BSA and 0.23 mM pyruvate. COCS were incubated at 37 C in 5% CO2 in microwells (96-well plates; Nunc, Roskilde, Denmark) in 100 µl DMEM supplemented with 2% charcoal-stripped FCS (sFCS) (24) and 0.23 mM pyruvate in the absence or presence of 100 ng/ml oFSH, T3 in the range of 0.1–100 nM, or oFSH plus T3. After 16–18 h, COCS were examined to evaluate the process of cumulus expansion. This analysis was based on the morphological observation of cumulus cell dispersion and embedding in the matrix containing hyaluronic acid. Afterward, to determine the stage of meiotic maturation, the cumulus-enclosed oocytes were deprived of surrounding cumulus cells either by simple pipetting or by a brief incubation in the presence of 0.1% hyaluronidase when cumuli were expanded. The oocytes that had reached metaphase II were identified by the presence of polar body.

Collection and culture of granulosa cells
Granulosa cells were collected after COCS removal, centrifuged, and resuspended in DMEM-2% sFCS to obtain a final concentration of 2–3 x 106 viable cells/ml, as judged by the trypan blue dye exclusion test. Two hundred microliters of this cell suspension were seeded onto microwells and cultured for 24 h at 37 C in 5% CO2 in air. At the end of this incubation period, cells were washed twice and further incubated for 24 h in DMEM supplemented with 0.1% BSA and ITS (5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium). Cells were washed and treated for different periods of time (2, 4, and 6 days) in the absence (basal) or presence of oFSH (100 ng/ml), different doses of T3 (0.1–100 nM), or oFSH plus T3. In another series of experiments, cells were incubated for 6 days in the absence or presence of 0.1 mM cAMP or 1 mM MIX, with or without 100 nM T3. Media were changed every other day. To all cultures 19OH-androstenedione (1 µM), as androgenic substrate for aromatase activity, was added 16 h before the end of hormonal treatments. Afterward, culture media were collected, centrifuged to remove cellular debris, and stored at -70 C until analyzed for E2 production. Total cell protein content was measured according to the method of Bradford (25).

E2 determination
E2 released by granulosa cells cultured in the different experimental conditions was determined by RIA as previously described (26). A 1:10,000 dilution of E2 antiserum (200 µg), 50-µl aliquots of sample media, or increasing concentrations of E2 (standard) and 200 µl (15,000 cpm) of [3H]E2 were incubated for 16–20 h at 4 C. At the end of the incubation time, 200 µl charcoal-dextran solution (0.2% Norit-A charcoal and 0.25% Dextran T-70) in PBS were added to each tube. After 10-min incubation at 4 C, tubes were centrifuged for 10 min at 3,000 rpm. The supernatant was decanted into 20-ml scintillation vials, and 15 ml Pico-Fluor 40 (Packard, Milan, Italy) were added to each vial. Radioactivity was counted in a Beckman Coulter, Inc. LS-7500 counter (Fullerton, CA). Results are expressed as picograms of E2 per µg cell proteins.

RNA isolation and analysis
Total cellular RNA was extracted from cultured granulosa cells treated with or without oFSH (100 ng/ml) in the presence or absence of T3 (100 nM) for 6 days using the acid guanidinium thiocyanate-phenol-chloroform method (27). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. Levels of aromatase mRNA were determined by the RT-PCR method. Total RNA (0.5 µg) were reverse transcribed, in a final volume of 20 µl, using 200 U cloned M-MLV reverse transcriptase in the presence of 2.5 µM random hexamers and 1 mM deoxy-NTP for 1 h at 37 C, then heat denatured for 5 min at 95 C; for each sample half of the resulting complementary DNA was subjected to PCR using primers designed for the amplification of either aromatase (upstream, 5'-GCACGAGAATGGCATCAT-3'; downstream, 5'-GTTAGAAGTGTCCAGCATG-3'; amplified product, 200 bp) (28) or the cyclophilin housekeeping gene (upstream, 5'-AGAAGCGCATGAGCATTGTGGAAG-3'; downstream, 5'-TGCTCTCCTGAGCTACAGAAGGAA-3'; amplified product, 159 bp) (29). Controls for DNA contamination or PCR carryover were performed omitting the M-MLV or the RNA during RT. Complementary DNAs were amplified using a DNA thermal cycler (Perkin Elmer/Cetus) and the Taq DNA polymerase (2 U/tube) with 15 pmol of both upstream and downstream primers and 2.2 mM magnesium chloride in a final volume of 50 µl that was overlaid with 25 µl mineral oil. Thirty-five cycles (60 sec at 94 C, 60 sec at 60 C, and 60 sec at 72 C with a 10-min final extension) were applied; in these conditions preliminary experiments demonstrated that the plateau for the housekeeping amplification was not reached. For each sample 25 µl PCR amplification product were analyzed on 2% agarose gel and stained with ethidium bromide. Standard DNA mol wt (Ladder VI, Boehringer Mannheim, Indianapolis, IN) was run to provide the appropriate size marker. Amplified products were quantitated by Flour-S MultiImagex (Bio-Rad Laboratories, Inc.), and aromatase mRNA levels were normalized against cyclophilin.

Statistical analysis
Each data point represents the mean ± SEM of three experiments unless otherwise specified. Thyroid hormone dose-response curves were analyzed by curve fitting with four-parameter logistic functions using the Allfit computer program (30). Data were analyzed by Student’s t test using the STATPAC computer program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cumulus complex expansion and oocyte meiotic maturation
To determine the effects of T3 on basal or oFSH-induced cumulus expansion, COCS isolated from immature or adult ovaries were cultured for 16–18 h in the absence or presence of oFSH (100 ng/ml), different concentrations of T3 (0.1–100 nM), and oFSH plus T3. The results reported in Fig. 1Go and Table 1Go showed that T3 (100 nM), either alone or together with oFSH, did not interfere with the in vitro process of cumulus expansion in immature mice. Also, oocyte meiotic maturation up to metaphase II stage was unaffected by T3, as more than 90% of the in vitro matured COCS underwent polar body emission regardless of the hormonal treatment (Table 1Go). Lower T3 concentrations gave identical results (data not shown). When the same experimental protocols were used to evaluate cumulus expansion and oocyte meiotic maturation of COCs obtained from adult mice, we found no effects of T3 (0.1–100 nM) on either processes (data not shown).



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  		Figure 1. In vitro effect of T3 on basal and FSH-induced expansion of cumuli oophori isolated from immature mice. COCS were cultured in the absence (basal) or presence of T3 (100 nM), oFSH (100 ng/ml), or oFSH plus T3 for 16–18 h as described in Materials and Methods. Magnification, x200.

 

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  		Table 1. In vitro T3 effects on basal and FSH-induced cumulus cell-oocyte complex (COCS) expansion and oocyte meiotic maturation up to metaphase 2 stage, evidenced by polar body (PB) emission, from immature mice

 
Effect of T3 on aromatase activity of cultured granulosa cells
The effects of T3 on basal and oFSH-induced aromatase activity were evaluated by measuring E2 levels released by cultured granulosa cells. As evidenced in Fig. 2Go, the basal aromatase activity of granulosa cells, harvested from immature mice and cultured for 6 days, was not affected by T3 (100 nM) treatment. oFSH (100 ng/ml) strongly stimulated E2 release from 3.8 ± 2.8 to 62.9 ± 2.9 pg/µg protein (P < 0.01). FSH stimulation of aromatase activity was inhibited by T3 cotreatment in a dose-dependent manner, with an ED50 of 0.87 ± 0.21 nM (Fig. 2Go). The effect of T3 on oFSH-induced aromatase activity was time dependent. Indeed, as shown in Fig. 3Go, the inhibitory effect of T3 became fully appreciable (P < 0.01) only after 6 days of hormonal treatment, whereas at the earlier time points it was absent (2 days) or barely detectable (4 days).



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  		Figure 2. In vitro dose-dependent effect of T3 on FSH-stimulated activity in immature granulosa cells. Immature mice granulosa cells were cultured in DMEM containing 2% sFCS for 24 h. The next day, cells were washed twice and incubated for an additional 24 h in DMEM supplemented with 0.1% BSA and ITS. Afterward, cells were incubated for 6 days in the absence or presence of oFSH (100 ng/ml) and oFSH plus T3 at different doses (0.1–100 nM). Media were changed every other day. 19OH-androstenedione was added 16 h before the end of the hormonal treatments. Culture media were then collected and analyzed for E2 production by RIA. Each point represents the mean ± SEM of triplicate wells representative of two independent experiments. *, FSH plus 10-8 M T3 and FSH plus 10-7 M T3 vs. FSH, P < 0.01.

 


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  		Figure 3. In vitro time-dependent effects of T3, FSH, and FSH plus T3 on immature granulosa cell aromatase activity. Granulosa cells were cultured in DMEM containing 2% sFCS for 24 h, washed, and incubated for an additional 24 h in DMEM supplemented with 0.1% BSA and ITS. The cells were then incubated for different periods of time (2, 4, and 6 days) in the absence or presence of oFSH (100 ng/ml), T3 (100 nM), and FSH plus T3, with 19OH-androstenedione added 16 h before the end of the hormonal treatments. Culture media were collected and analyzed for E2 production by RIA. Data pointsrepresent the mean ± SEM of triplicate wells of one of two similar experiments. *, P < 0.01.

 
To determine whether the inhibitory effect of T3 was due to an impairment of cAMP accumulation after oFSH treatment, granulosa cells were incubated for 6 days in the absence or presence of (Bu)2cAMP, with or without T3. As shown in Fig. 4AGo, basal E2 release was stimulated by (Bu)2cAMP (0.1 mM; P < 0.01). This stimulation was significantly inhibited by coincubation with 100 nM T3 (P < 0.01). In parallel experiments, the possibility that the inhibitory action of T3 could be mediated by a stimulation of cAMP-phosphodiesterase (PDE) activity was also evaluated. As shown in Fig. 4BGo, the PDE inhibitor MIX (1 mM) significantly (P < 0.01) induced basal aromatase activity in cultured granulosa cells, but this stimulation was once again abolished after cotreatment with 100 nM T3 (MIX plus T3 vs. MIX, P < 0.01).



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  		Figure 4. In vitro inhibitory effects of T3 on (Bu)2cAMP (A)- and MIX (B)-stimulated aromatase activity in immature granulosa cells. Granulosa cells were cultured in DMEM containing 2% sFCS for 24 h, washed twice, and incubated for an additional 24 h in DMEM supplemented with 0.1% BSA and ITS. Cells were then washed and incubated for 6 days in the absence or presence of (Bu)2cAMP (0.1 mM), T3 (100 nM), or (Bu)2cAMP plus T3 (A); or MIX (1 mM), T3 (100 nM), or MIX plus T3 (B). 19OH-androstenedione was added 16 h before the end of the hormonal treatments. Culture media were then collected and analyzed for E2 production by RIA. Each point represents the mean ± SEM of three experiments. *, (Bu)2cAMP plus T3vs. (Bu)2cAMP and MIX plus T3vs. MIX, P < 0.01.

 
The same inhibitory action of T3 could be replicated in similar experiments using granulosa cells obtained from adult mice (data not shown). However, with respect to the findings mentioned above, the only major difference was in the time course of the effect. In fact, the inhibitory action of T3 (100 nM) on oFSH-induced aromatase activity was already evident (P < 0.01) after 2 days of treatment, as shown in Fig. 5Go.



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  		Figure 5. In vitro effect of T3 on adult granulosa cell aromatase activity. Granulosa cells were cultured in DMEM containing 2% sFCS for 24 h, washed, and incubated for an additional 24 h in DMEM supplemented with 0.1% BSA and ITS. Cells were then incubated for 2 days in the absence or presence oFSH (100 ng/ml), T3 (100 nM), or FSH plus T3. 19OH-androstenedione was added to the culture media 16 h before the end of the hormonal treatments. Culture media were then collected and analyzed for E2 production by RIA. Each point represents the mean ± SEM of three experiments. *, FSH plus T3vs. FSH, P < 0.01.

 
Finally, the effects of T3 (100 nM) on oFSH (100 ng/ml) modulation of aromatase mRNA levels in granulosa cells cultured for 6 days were assessed by semiquantitative RT-PCR (Fig. 6Go). Densitometric analysis of the amplified product showed that FSH increased the level of aromatase mRNA by 2.4 ± 0.5-fold. This stimulation was completely abolished by cotreatment of cultured granulosa cells with T3 (Fig. 6Go).



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  		Figure 6. Thyroid hormone effect on FSH-induced aromatase mRNA level in cultured immature granulosa cells. Total RNA was extracted from cultured granulosa cells and treated for 6 days with or without FSH (100 µg/ml) in the absence or presence of T3 (100 nM), as described in Materials and Methods section. Aromatase and cyclophilin mRNAs levels were assessed by RT-PCR using 0.5 µg total RNAs for each sample as described in Materials and Methods. Amplification products were separated on 2% agarose gel and stained with ethidium bromide. The data shown are representative of three different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although in mammals the development of the female reproductive system during fetal and early postnatal life appears to be T3 independent (9, 13), an increasing amount of evidence indicates an important role for T3 in adult female reproductive functions (8, 9, 10, 11, 12, 13). Indeed, changes in T3 levels are associated with menstrual disturbance and impaired fertility (9, 13). Even if these effects are due at least in part to an altered secretion of pituitary gonadotropins after T3 hypo- or hypersecretion (10, 11, 12), works from different laboratories have suggested a direct role of T3 in ovarian functions. In particular, the presence of T3 receptors (TR) in mammalian granulosa cells (17, 18, 19, 20, 21, 22), which appear to be functionally coupled to the modulation of gonadotropin-induced steroidogenesis (14, 15, 16, 17, 31), has been shown. More recently, the presence of TR has also been described in human cumulus cells and oocytes (23), suggesting a possible involvement of T3 in physiological processes leading to the production of mature and fertilizable eggs.

The results of the present study demonstrated, however, that two key processes required for adequate cumulus cell-oocyte complex maturation, such as cumulus expansion and oocyte meiotic resumption and progression up to metaphase II stage, are unaffected by in vitro T3 treatment in both immature and adult female mice. Nevertheless, these findings do not exclude the possible modulation by T3 of other oocyte functions needed for successful fertilization and/or further embryo development. Alternatively, it may be possible that oocyte TR mRNAs and the related proteins are stored during oogenesis to become functional only later during development. This appears to be the case of the amphibian Xenopus laevis oocytes, in which high levels of maternally inherited TR{alpha} mRNA and protein have been described, despite the fact that the competence of the larvae to respond to T3 is only acquired late in embryonic development (32, 33). Lastly, a ligand-independent function for TR protein during oocyte and/or early embryonic development may be hypothesized, considering the ability of TR to function as a negative modulator of transcription (34, 35, 36).

It is well established that steroid hormones released by granulosa cells are critical for proper follicle as well as endometrium development (1, 2, 3, 4, 37). Our results demonstrated that T3 induces a drastic reduction of FSH-stimulated aromatase activity in cultured granulosa cells obtained from both immature and adult mice. This inhibitory action is time and dose dependent, with the half-maximally effective dose of T3 being 0.87 nM. This value is in agreement with the reported values of the TR dissociation constant (Kd) ranging from 0.4–5.5 nM in granulosa cells of different mammalian species (17, 19, 21, 38). It is important to note that in immature granulosa cells this inhibitory action was only evident after 6 days of hormonal treatment, whereas in granulosa cells derived from adult ovaries a significant inhibition by T3 of aromatase activity was present after 2 days of culture. At the moment, the reason(s) for the higher sensitivity to T3 shown by adult vs. immature mouse granulosa cells is unknown. One possibility is that TR are differentially expressed in immature cells with respect to adult granulosa cells. This hypothesis is currently under investigation.

The ability of T3 to modulate cAMP-PDE activity in brain and liver cells has been documented (39, 40). More interestingly, T3 has been shown to augment hCG-induced cAMP accumulation in human luteinized granulosa cells through a possible inhibitory effect on PDE activity (17). From our results, it appears clear that the inhibitory action of T3 on FSH-stimulated aromatase activity in granulosa cells is beyond cAMP accumulation, as demonstrated by the fact that both (Bu)2cAMP- and MIX-stimulated E2 releases are significantly decreased after T3 cotreatment of cultures.

This was further supported by the ability of T3 to inhibit FSH-induced aromatase mRNA levels in cultured granulosa cells as judged by semiquantitative RT-PCR. It remains, however, to be established whether T3 acts directly on the transcription of the aromatase gene or whether it is able to affect the stability of its mRNA.

Several reports have shown the ability of T3 to modulate gonadotropin-induced steroidogenic activity in cultured granulosa cells. However, the reported results appear to be quite controversial in the different animal species. In fact, in mammalian granulosa cells T3 has been reported to inhibit (14, 17), to increase (15, 16), or to affect in a follicle size-dependent manner (15, 31) gonadotropin-induced steroidogenesis. One reason for these discordant data could be the different responsiveness to T3 of granulosa cells isolated from follicles at different stages of antral development. Indeed, Maruo and colleagues (38) demonstrated that porcine granulosa cells isolated from small and medium follicles possess a higher number of T3-binding sites with respect to those present in granulosa cells isolated from large antral follicles. In addition, the different animal species analyzed and/or the different culture conditions used may be responsible for the discordant observations. It is worth noting that Wakim and colleagues (16) suggested that the stimulatory effects of T3 on mammalian granulosa cells steroidogenesis are dependent upon the presence of insulin in the culture medium. In the present study, however, T3 does not affect basal, whereas it inhibits FSH-induced, aromatase activity of cultured mouse granulosa cells despite the presence of insulin in the culture medium. Interestingly, our in vitro data appear to corroborate those recently reported by Tamura and colleagues showing an in vivo inhibitory role for thyroid hormone on ovarian aromatase activity and inhibin expression (41). Indeed, they demonstrated that thyroidectomy in immature female rats causes an increased secretion of inhibin and estradiol during ovarian development induced by the administration of equine CG and that the high plasma levels of both ovarian hormones could be recovered to control levels by T4 treatment.

Lastly, it may be of interest to note that in Sertoli cells, the male counterpart of granulosa cells, an inhibition of aromatase activity by thyroid hormone, of the same order of magnitude as that demonstrated here, has been described in vitro and in vivo (26, 42, 43).

In conclusion, the inhibitory effect of T3 on FSH-induced aromatase activity herein described can be regarded as a part of the complex multihormonal regulation of mammalian follicle development and may contribute to explain the alterations in female reproductive functions after T3 hypo- or hypersecretion.


   Acknowledgments
 
We are very grateful to NIDDK’s National Hormone and Pituitary Program and Dr. A. F. Parlow (NIDDK, Bethesda, MD) for providing us with ovine FSH, and to Dr. G. Melillo (Dompè Laboratory, L’Aquila, Italy) for the assistance with cell cultures. We thank Ms. D. Di Gregorio and Ms. P. Minelli for preparation of the manuscript. We are particularly grateful to Dr. J. R. Tata (National Institute for Medical Research, London, UK) for providing us with T3, and to Mrs. E. Heather for proofreading the manuscript.


   Footnotes
 
1 This work was supported by MURST 40–60% grants. Back

Received July 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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