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Thyroid Hormone Regulates the Cell Cycle Inhibitor p27^Kip1 in Postnatal Murine Sertoli Cells

Department of Veterinary Biosciences (D.R.H., P.S.C.) and Division of Nutritional Sciences (P.S.C.), University of Illinois-Urbana, Urbana, Illinois 61802; and Department of Molecular Genetics (S.J., H.K.), University of Illinois-Chicago, Chicago, Illinois 60607

Address all correspondence and requests for reprints to: Paul S. Cooke, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802. E-mail: p-cooke{at}uiuc.edu.

	Abstract

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Thyroid hormone regulates early postnatal Sertoli cell proliferation. Transient neonatal hypothyroidism allows prolonged postnatal Sertoli cell mitogenesis and doubles adult Sertoli cell numbers, testis weight, and sperm production. The mechanism of this effect is unknown. Cell proliferation is stimulated by cyclins and cyclin-dependent kinases and inhibited by cyclin-dependent kinase inhibitors (CDKIs). T₃ regulates the CDKI p27^Kip1 in other cell types, and mice lacking p27^Kip1 have increased testis size. To test the hypothesis that T₃ regulates Sertoli cell mitogenesis by acting through p27^Kip1, we compared expression of p27^Kip1 in Sertoli cells of testes from euthyroid, hypothyroid, and hyperthyroid mice. At postnatal d 5–25, testes were collected and immunostained for p27^Kip1 expression, or Sertoli cells were isolated enzymatically and used for p27^Kip1 Western blotting. p27^Kip1 immunostaining was low in rapidly proliferating 5-d-old Sertoli cells but had increased strongly in nonproliferating 25-d-old Sertoli cells. p27^Kip1 immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 d, consistent with increased Sertoli cell proliferation in these mice. Western blotting corroborated the p27^Kip1 immunostaining, and p27^Kip1 expression was greater in Sertoli cells from control compared with hypothyroid mice at postnatal d 10 and 16, but p27^Kip1 expression was comparable by d 25. Hyperthyroidism increased p27^Kip1 immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-d-old hyperthyroid mice expressed more p27^Kip1 than control mice. These results indicate that thyroid hormone status affects p27^Kip1 expression in neonatal Sertoli cells, suggesting that T₃ effects on Sertoli cell proliferation may be mediated through this CDKI.

	Introduction

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SERTOLI CELLS IN mice and rats are proliferating rapidly at birth, but mitogenesis declines during the neonatal period and ceases by about postnatal d 15 (1, 2). Termination of Sertoli cell proliferation is accompanied by differentiation of these cells and increased production of proteins characteristic of the mature Sertoli cell. FSH is the major Sertoli cell mitogen (3, 4, 5), but the decrease and eventual cessation in Sertoli cell proliferation despite continual neonatal FSH exposure (6) suggests that other factors regulate the transition of Sertoli cells from the mitotic to the postmitotic state during early postnatal development. Each Sertoli cell supports a fixed number of germ cells, so Sertoli cell number is the major factor that establishes the magnitude of sperm production. Therefore, understanding factors regulating the proliferation and establishment of the final adult Sertoli cell population is one of the most critical questions in testicular biology.

Thyroid hormone plays an integral role in Sertoli cell development, and it may be responsible for developmental changes that finally eliminate the mitogenic response to FSH. The neonatal testis expresses high levels of thyroid hormone receptors, predominantly in Sertoli cells (7, 8, 9, 10), and thyroid hormone levels peak around 2 wk postnatally in the mouse (11), thus inferring a direct effect of thyroid hormone on early postnatal Sertoli cells. Transient neonatal hypothyroidism in rats results in doubling of adult testis size, Sertoli cell number, and sperm production (12, 13, 14), and similar effects are seen in mice (1). This reflects a lengthened period of Sertoli cell proliferation in hypothyroid rats and mice (1, 15). These hypothyroid effects are directly on Sertoli cells, as shown by the decrease in proliferation and increased expression of Sertoli cell differentiation markers in neonatal Sertoli cells treated with T₃ in vitro (8, 16, 17). Similarly, administration of exogenous T₃ to neonatal rats results in a precocious cessation of Sertoli cell proliferation and accelerated maturation as shown by premature tubular lumen formation (15); the latter may reflect stimulatory effects of T₃ on the gap junction protein connexin 43 (18).

The molecular mechanism by which T₃ induces cessation of proliferation and exit from the cell cycle in Sertoli cells is unknown. Cell cycle progression is regulated by cyclins and cyclin-dependent kinases; these in turn are regulated by specific cyclin-dependent kinase inhibitors (CDKIs) such as the Kip/Cip and INK4 families. Knockouts of the Kip/Cip family member p27^Kip1 have been developed (19, 20, 21), and testis size is doubled in these mice, thus suggesting a possible link between p27^Kip1 expression and Sertoli cell proliferation. In this report, we present data indicating that T₃ may act through p27^Kip1 to induce its effects on Sertoli cell proliferation. Specifically, neonatal hypothyroidism reduces p27^Kip1 levels in Sertoli cells, thereby allowing for continued cell cycle progression and extended Sertoli cell proliferation, whereas hyperthyroidism produces opposite effects. Therefore, T₃ effects on p27^Kip1 may be the critical mechanism by which this hormone induces cessation of Sertoli cell proliferation, and this result in concert with other recent literature suggest that T₃ effects on p27^Kip1 may be the general mechanism by which this hormone regulates cell proliferation in T₃-responsive tissues.

	Materials and Methods

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Animals and treatments
C57BL/6 mice were bred in our colony and maintained as described (1). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois, and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Litters were randomly assigned to euthyroid, hypothyroid, or hyperthyroid groups. Pups were made hypothyroid by the addition of 0.1% 6-propyl-2-thiouracil (PTU) to the dam’s drinking water, as described (1). PTU-treated water contained cherry Kool-Aid to increase palatability. Drinking water for the euthyroid litters was untreated. To make pups hyperthyroid, T₃ (Sigma, St. Louis, MO) was dissolved in 0.025 N sodium hydroxide then diluted in physiological saline. T₃ was administered by daily subcutaneous injections of 100 µg/kg BW in 10 µl of vehicle (15).

Sertoli cell isolation, immunohistochemistry, and Western blotting
Testes were dissected from mice at postnatal d 5, 10, 16, and 25 (day of birth = 0). Sertoli cells were isolated by sequential enzymatic digestion (22) as previously modified (23). Testes were weighed and minced, then incubated in 2 ml of enzyme solution per 100 mg of tissue. Sertoli cell protein was extracted in IGEPAL CA-630 (Sigma) lysis buffer (19), with vortexing and two freeze-thaw cycles to disrupt the cell membranes. Total protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL) as per manufacturer’s directions.

For p27^Kip1 immunohistochemistry, testes were fixed overnight in neutral buffered formalin at room temperature before dehydration and paraffin embedding. Tissues were sectioned at 4 µm and then deparaffinized and rehydrated. Endogenous peroxidase activity was quenched by incubating sections in 0.3% H₂O₂ for 30 min. To optimize p27^Kip1 detection, slides were placed in boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min, then allowed to cool to room temperature. Immunodetection of p27^Kip1 protein was performed using a murine mononclonal IgG1 to mouse p27^Kip1 (BD Transduction Laboratories, Lexington, KY). Binding of primary antibody was localized by using the horseradish peroxidase-Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine substrate kit (Vector) according to the supplier’s instructions. Negative control tissue sections were processed with normal goat serum instead of the primary antibody to determine nonspecific staining. Following immunostaining, some tissues were counterstained with Gill’s formulation no. 1 hematoxylin (Fisher Scientific, Pittsburgh, PA).

The regulation of p27^Kip1 has been reported to involve posttranscriptional mechanisms, so p27^Kip1 protein levels were examined by Western blotting. Sertoli cell lysates (30 µg total protein) were electrophoresed on a discontinuous 12.5% SDS-PAGE (Criterion Gel, Bio-Rad, Hercules, CA) using the buffer system of Laemmli (24), then transferred onto 0.45 µm polyvinylidene difluoride membrane. Nonspecific binding of protein was blocked by submerging the membrane in 5% (wt/vol) nonfat dry milk in PBS (pH 7.4) for 1 h with moderate shaking. The p27^Kip1 protein was detected using the same primary antibody as described above. Immunoreactive bands were detected using horseradish peroxidase-conjugated goat antimouse Ig (heavy and light) (Pierce) and SuperSignal West Pico Chemiluminescent Substrate (Pierce). Chemiluminescence was captured on autoradiographic film, and densitometrically analyzed using Quantity One software (Bio-Rad). Membranes were stained with Ponceau S (Sigma) to verify even transfer and equal loading of protein. p27^Kip1 expression was normalized to a family of 60-kDa proteins detected by Ponceau S staining. HeLa cell extracts provided with the primary antibody were used as a positive control.

Data analysis
All data are presented as mean ± SEM. Results were analyzed using either Student’s t test or one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test. Differences were considered significant at P < 0.05.

	Results

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Effects of hypothyroidism and hyperthyroidism on p27^Kip1 immunostaining
We examined postnatal testes from euthyroid, hypothyroid, and hyperthyroid mice at 5, 10, 16, and 25 d for p27^Kip1 protein expression using immunostaining. Germ cells did not stain at any time point in any of the groups (Fig. 1A), confirming previous work that p27^Kip1 staining in seminiferous tubules at this age is confined to Sertoli cells (25). p27^Kip1 staining was minimal in Sertoli cells of euthyroid mice at d 5. In contrast, Sertoli cell p27^Kip1 staining was more heterogeneous in testes from 5-d-old hyperthyroid mice, with some Sertoli cells showing clear p27^Kip1 immunostaining, whereas other Sertoli cells showed minimal staining (Fig. 1B), indicating that a portion of Sertoli cells from hyperthyroid mice were expressing significant levels of p27^Kip1 and may have already exited the cell cycle.

View larger version (106K): [in this window] [in a new window]   FIG. 1. Immunohistochemical detection of p27Kip1 in developing euthyroid, hyperthyroid, and hypothyroid testes. In 5-d-old testes, Sertoli cells (arrows) from euthyroid mice (A) showed minimal p27Kip1 staining, whereas Sertoli cells from hyperthyroid mice (B) stained more heavily, although a clear heterogeneity of Sertoli cell staining was seen in these mice. Germ cells (arrowheads) were unstained for p27Kip1 at all ages and with all treatments. Intense interstitial staining (asterisk) was present in testes from 5 d euthyroid and hyperthyroid mice. At 10 d of age, Sertoli cells from euthyroid mice (C) showed modest staining; Sertoli cell staining in hyperthyroid mice (D) appeared to be increased, whereas Sertoli cell staining was clearly reduced in testes of hypothyroid mice (E). Negative control sections stained without the primary antibody showed minimal background staining (F). At 16 d of age, Sertoli cell staining remained heavier in euthyroid mice (G) compared with hypothyroid mice (H). In all cases, samples from the same age were stained under identical conditions in the same staining run, and thus should be directly comparable.

Clear differences between p27^Kip1 expression in Sertoli cells of euthyroid, hypothyroid, and hyperthyroid mice were observed by d 10. Sertoli cells from 10-d-old euthyroid control mice displayed regular nuclear staining (Fig. 1C), whereas Sertoli cells from 10-d hypothyroid testes exhibited less intense diffuse nuclear staining, with the predominance of p27^Kip1 staining seen in the cytoplasm, where it created a halo around the nucleus (Fig. 1E). In contrast, immunostaining for p27^Kip1 was heavy in Sertoli cell nuclei of hyperthyroid testes (Fig. 1D) and appeared to be more intense compared with euthyroid controls.

At d 16, the disparity in Sertoli cell p27^Kip1 immunostaining in hypothyroid and euthyroid mice remained pronounced. Specifically, Sertoli cell nuclei from hypothyroid animals were lightly stained for p27^Kip1, whereas 16-d-old control euthyroid testes showed an overall increase in concentrated nuclear staining of Sertoli cells (Fig. 1, G and H). No observable differences were seen in p27^Kip1 expression between Sertoli cells from 16-d-old hyperthyroid and euthyroid mice (not shown). As expected, nuclear staining for p27^Kip1 in Sertoli cells was not different in hypothyroid compared with euthyroid mice at 25 d (data not shown), a time point where proliferation has ceased in Sertoli cells from euthyroid and hypothyroid mice. Negative controls where the primary antibody was omitted during the staining procedure showed minimal staining (Fig. 1F).

Western blot analysis of p27^Kip1 expression in postnatal Sertoli cells
Sertoli cells were isolated from euthyroid, hypothyroid, and hyperthyroid neonatal mice at postnatal d 5, 10, 16, and 25. Sertoli cell lysates were used to detect relative differences in p27^Kip1 expression by Western blot after normalizing p27^Kip1 expression to an internal loading control. p27^Kip1 protein expression increased temporally between postnatal d 5 and 25 (data not shown). Despite the differences in p27^Kip1 immunostaining, Western blotting did not detect a significant difference between p27^Kip1 expression in Sertoli cells from euthyroid and hyperthyroid mice at d 5 (data not shown), most likely due to relatively modest changes seen in p27^Kip1 in the hyperthyroid group at this age. Conversely, at postnatal d 10, when Sertoli cells from hyperthyroid mice are approaching cessation of proliferation, normalized p27^Kip1 expression was significantly higher (P < 0.01) in Sertoli cell lysates from these hyperthyroid mice (6.9 ± 1.6; n = 6) compared with euthyroid controls (3.1 ± 0.4; n = 7) (Fig. 2). p27^Kip1 expression in Sertoli cell lysates from 10-d euthyroid testes was not significantly different from that in Sertoli cells from hypothyroid mice (2.1 ± 0.4; n = 7); the 30% decrease in p27^Kip1 in the latter did not reach statistical significance, but there was a strong trend toward a decrease in hypothyroid vs. euthyroid mice (P = 0.07). p27^Kip1 expression in Sertoli cells from hyperthyroid mice was approximately 3.3-fold greater than that in Sertoli cells from hypothyroid mice (P < 0.01).

View larger version (34K): [in this window] [in a new window]   FIG. 2. Western analysis of p27Kip1 expression in Sertoli cells from 10-d-old testes. A, Immunodetection of p27Kip1 expression in Sertoli cells from euthyroid, hypothyroid, and hyperthyroid mice. Each lane represents Sertoli cell lysates from individual mice. The protein recognized by the antibody was the correct relative molecular mass of 27,000, as determined by molecular mass markers and use of HeLa cell lysates provided by the supplier of the p27Kip1 antibody that served as a positive control. B, Densitometric analysis of p27Kip1 expression in Sertoli cells from euthyroid, hypothyroid and hyperthyroid mice. Membranes were stained with Ponceau S to verify even transfer and equal loading of protein, then p27Kip1 expression was normalized to a family of 60-kDa proteins detected by Ponceau S staining and quantitated. All groups were significantly different from each other (P < 0.05), with the exception that the hypothyroid group showed a strong trend for decreased p27Kip1 levels relative to the euthyroid group (P = 0.07), but this difference did not reach statistical significance. Data are shown as mean ± SEM (n = 6–7 for each group).

View larger version (31K): [in this window] [in a new window]   FIG. 3. Western analysis of p27Kip1 expression in Sertoli cells from 16-d-old testes. A, Immunodetection of p27Kip1 expression in Sertoli cells from euthyroid and hypothyroid mice. Each lane represents Sertoli cell lysates from individual mice. B, Densitometric analysis of p27Kip1 expression in Sertoli cells from euthyroid and hypothyroid mice. p27Kip1 expression was normalized and quantitated as described in Fig. 2. The euthyroid and hypothyroid groups were significantly different from each other (P < 0.01). Data are shown as mean ± SEM, and n = 9 for each group.

View larger version (38K): [in this window] [in a new window]   FIG. 4. Western analysis of p27Kip1 expression in Sertoli cells from 25-d-old testes. A, Immunodetection of p27Kip1 expression in Sertoli cells from euthyroid and hypothyroid mice. Each lane represents Sertoli cell lysates from individual mice. B, Densitometric analysis of p27Kip1 expression in Sertoli cells from euthyroid and hypothyroid mice. p27Kip1 expression was normalized and quantitated as described in Fig. 2. The euthyroid and hypothyroid groups were not stastistically different from each other (P > 0.05) at d 25. Data are shown as mean ± SEM (n = 12 for each group).

	Discussion

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p27^Kip1 is expressed at high levels in testis (19), and the testicular expression predominantly reflects p27^Kip1 expression in Sertoli cells, suggesting a potentially important role for this protein in Sertoli cells. Expression of p27^Kip1 is low in rapidly proliferating neonatal rat Sertoli cells but is high in postmitotic adult Sertoli cells (25), indicating that p27^Kip1 may be a critical negative regulator of Sertoli cell proliferation. This putative link between p27^Kip1 and Sertoli cell proliferation is consistent with the low p27^Kip1 expression in Sertoli cell tumors (28) whose cells are proliferating rapidly, whereas neighboring normal Sertoli cells are not proliferating and express high p27^Kip1 levels.

The molecular mechanism by which thyroid hormone decreases Sertoli cell proliferation has been unknown. However, a common phenotype, testicular organomegaly, is observed with the transient neonatal hypothyroidism model and the p27^Kip1 knockout mouse, thus suggesting a potential common developmental pathway. Mice allowed to recover from transient neonatal hypothyroidism have increased adult testis size, Sertoli cell number, and sperm production (1). Similarly, testis size in p27^Kip1 knockout mice is twice that of wild-type littermates (19), and these knockouts have normal spermatogenesis (25). Importantly, we recently noted large increases in Sertoli cell number and sperm production in p27^Kip1 knockout mice (our unpublished data). Knockouts of all other CDKIs have been developed. With the exception of p18^Ink4c knockouts, which exhibit similar gigantism and organomegaly although their testes are affected to a lesser extent (29), the testicular organomegaly seen in the p27^Kip1 knockout mice is not seen with other CDKI knockouts (reviewed in Ref. 30), further suggesting p27^Kip1 as a potential target of thyroid hormone’s actions.

Our results indicate that thyroid hormone status affects p27^Kip1 expression in neonatal Sertoli cells, with hypothyroidism leading to reduced p27^Kip1 expression during the critical period of Sertoli cell proliferation. p27^Kip1 protein levels quantified by Western blotting corroborated testicular immunohistochemical staining. Differences in p27^Kip1 levels observed in hypothyroid neonatal testes correlate well with the increased Sertoli cell proliferation in hypothyroid mice during this same period (1).

Based on the critical role of p27^Kip1 in cell cycle progression and the ability of hypothyroidism to delay the normal postnatal increase in this protein, we postulate that T₃ can regulate p27^Kip1 in Sertoli cells and that this may be critical for extended Sertoli cell proliferation induced by hypothyroidism. This suggests that the normal role of T₃ in neonatal testes is to increase p27^Kip1 expression, thereby decreasing Sertoli cell proliferation. Suppression of p27^Kip1 in hypothyroid neonates allows for extended Sertoli cell proliferation and ultimate increases in adult testis size, Sertoli cell number, and sperm production. Furthermore, our results and previous results (25, 28) showing an inverse relationship between Sertoli cell p27^Kip1 expression and proliferation strongly suggest that the testicular organomegaly seen in p27^Kip1 knockout mice (19) is primarily a result of increased Sertoli cell proliferation and consequent increases in adult Sertoli cell populations.

Neonatal hyperthyroidism causes premature cessation of Sertoli cell proliferation (15). If our hypothesis that thyroid hormone affects Sertoli cell proliferation through effects on p27^Kip1 is correct, hyperthyroidism would be expected to increase p27^Kip1 expression. Hyperthyroidism produces Sertoli cell p27^Kip1 expression levels over 3-fold greater than those seen in hypothyroid mice and provides further evidence that T₃ regulates Sertoli cell p27^Kip1.

Despite the ability of hypothyroidism to prolong Sertoli cell proliferation and delay the postnatal increase in p27^Kip1, these cells stop proliferating at approximately d 25 (1). Therefore, it would be expected that delayed Sertoli cell p27^Kip1 expression in hypothyroid mice should no longer be demonstrable by d 25 when proliferation in Sertoli cells from hypothyroid and euthyroid mice have both ceased. The similar expression of p27^Kip1 in 25-d-old Sertoli cells from hypothyroid and euthyroid mice confirms this and indicates that differences in p27^Kip1 expression induced by hypothyroidism are transient and no longer seen by d 25. These results are consistent with a central role for p27^Kip1 in Sertoli cell proliferation.

T₃ effects on p27^Kip1 expression levels have not been previously reported in Sertoli cells. Our present findings are in close agreement with those presented as a companion paper to this study that T₃ increases p27^Kip1 levels in cultured neonatal rat Sertoli cells (31). Testosterone and retinoic acid, which inhibit Sertoli cell proliferation, also increase p27^Kip1 expression levels (31), suggesting that modulation of p27^Kip1 expression may be a critical common mechanism by which various hormonal signals regulate Sertoli cell proliferation.

Although not previously recognized as such, this mechanism whereby T₃ increases p27^Kip1 levels may be common in developing organs. T₃ induces growth arrest and stimulates differentiation in the N2a-ß neuronal cell line concomitant with increased p27^Kip1 (32). Similar results have been observed in other T₃-responsive tissues, including oligodendrocytes (33), cardiac myocytes (34), and chondrocytes (35). Our data, combined with these previous findings, strongly suggest that stimulatory effects of T₃ on p27^Kip1 to induce cessation of cell proliferation during development are likely an important developmental mechanism for the maturational effects of thyroid hormone and its ability to regulate the ultimate size of various cell populations and organs.

The mechanism by which T₃ regulates p27^Kip1 in Sertoli cells is unclear but may involve both transcriptional and posttranscriptional affects. T₃ induces a 3-fold increase in p27^Kip1 mRNA in N2a-ß cells, suggesting a role for T₃ in transcriptional regulation of p27^Kip1 (32). However, no thyroid hormone-responsive element was found in the p27^Kip1 promoter (32), and T₃ does not increase p27^Kip1 mRNA in other systems (33, 35). p27^Kip1 protein half-life is lengthened in N2a-ß neuronal cultures containing T₃ (32), thus causing a build up of p27^Kip1 protein levels within these cells. This change may result from effects on the ubiquitin-proteosome pathway (36, 37). In addition, T₃ regulates testicular expression of connexin 43 (18), and in cell lines increases in connexin 43 have been shown to increase p27^Kip1 levels and inhibit cell proliferation through effects on ubiquitin-proteosome ligases (38). Recent evidence in the murine embryonic carcinoma cell line P19 has shown that liganded thyroid hormone receptor may regulate the key S-phase transcription factor E2F-1 (39), which could directly affect proliferation. Clearly, T₃ exerts effects on the cell cycle are complex and may involve multiple mechanisms that affect Sertoli cell proliferation.

T₃ synergizes with FSH to promote granulosa cell differentiation (40), and other results have shown that T₃ may regulate granulosa cell proliferation. Thyroid hormone receptor is expressed in granulosa cells (41), the female homolog of Sertoli cells. Recent results have indicated that proliferation is prolonged during differentiation of granulosa cells lacking p27^Kip1 (20, 42) and another member of the Cip/Kip family of CDKIs, p21^Cip1 (43). Based on the putative role of thyroid hormone in regulating p27^Kip1, it would appear plausible that T₃ actions on proliferation and differentiation in granulosa cells may also be mediated by effects on Cip/Kip proteins, and this area needs further investigation.

In summary, our results show that expression of the CDKI p27^Kip1 rises markedly over the neonatal period, increasing from low neonatally to high at d 25 in a pattern that is the inverse of the proliferative pattern of these cells. Hypothyroidism delays and hyperthyroidism advances the expression of p27^Kip1 in developing Sertoli cells. These results, in conjunction with previous results indicating an important role for p27^Kip1 in Sertoli cell proliferation, are consistent with the hypothesis that thyroid hormone modulates Sertoli cell proliferation through effects on p27^Kip1.

	Acknowledgments

 
The authors thank R. Hess (University of Illinois-Urbana) and L. Franca (Federal University of Minas-Gerais, Belo Horizonte, Brazil) for helpful discussions and reading of the manuscript, and the Histology Core of the National Institutes of Health-supported Center for Women’s Health and Reproduction (U54-HD 40093) for tissue processing. The authors thank S. Kiesewetter for technical assistance and M. Zakroczymski for assistance with figure preparation. The authors are grateful to J. Stark for his expertise and assistance with the Western blotting quantitation.

	Footnotes

 
This work was supported by NIH Grant HD38085 (to H.K.) and a Council on Food and Agricultural Research grant from the University of Illinois (to P.S.C.). D.H. was a recipient of a postdoctoral fellowship from the Reproductive Biology Research Training Program (NIH Grant T32 HD07028), University of Illinois-Urbana.

Abbreviations: CDKI, Cyclin-dependent kinase inhibitor; PTU, propylthiouracil.

Received March 26, 2003.

Accepted for publication April 30, 2003.

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