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BOR - Papers in Press, published online ahead of print April 27, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041426
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BIOLOGY OF REPRODUCTION 73, 396–403 (2005)
DOI: 10.1095/biolreprod.105.041426
© 2005 by the Society for the Study of Reproduction, Inc.

Regulation of Neonatal Sertoli Cell Development by Thyroid Hormone Receptor {alpha}11

Denise R. Holsberger 3, Sarah E. Kiesewetter 3, and Paul S. Cooke 2, 3,4 

Department of Veterinary Biosciences3 Division of Nutritional Sciences,4 University of Illinois,Urbana, Illinois 61802


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors {alpha}1 and ß1, which are commonly referred to as TR{alpha}1 and TRß1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TR{alpha}1 and TRß1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TR{alpha}KO), Thrb knockout (TRßKO), and wild-type (WT) mice. Triiodothyronine (T3) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRßKO mice versus that in their untreated controls, whereas T3 had a diminished effect on TR{alpha}KO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T3-treated WT and TRßKO testes. In contrast, T3 had no effect on these parameters in TR{alpha}KO mice. In untreated adult TR{alpha}KO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRßKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRßKO mice showed comparable increases following hypothyroidism. Our results show that TR{alpha}KO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRßKO mice, but not TR{alpha}KO mice, have normal Sertoli cell responsiveness to T3. Thus, effects of exogenous manipulation of T3 on neonatal Sertoli cell development are predominately mediated through TR{alpha}1.

Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the past 15 yr, numerous laboratories have shown that triiodothyronine (T3) plays a key role in Sertoli cell development [1–4] and, ultimately, affects the establishment of adult Sertoli cell populations and the magnitude of adult sperm production. Triiodothyronine has a direct inhibitory effect on Sertoli cell proliferation and a concomitant stimulatory effect on Sertoli cell differentiation. For example, transient neonatal hypothyroidism in rodents extends the length of Sertoli cell proliferation, leading to significant increases in Sertoli cell number, daily sperm production (DSP), and testis weight [1, 4–7]. Conversely, hyperthyroidism induced by exogenous T3 injections results in a premature cessation of mitogenesis and precocious canalization of seminiferous tubules [8].

Although the effects of T3 on the testis initially were described in rodents [1, 4–8], subsequent work has indicated that neonatal/juvenile hypothyroidism increases testis size in humans [9], roosters [10], and fish [11]. Similarly, high neonatal thyroid hormone levels in bulls resulted in decreased adult testis size and sperm production [12]. Clearly, the T3 effect on the developing testis is a critical process in mammalian and even nonmammalian species.

Effects of T3 on target organs are regulated by thyroid hormone receptors (TRs) that are nuclear ligand-modulated transcription factors encoded by two genes, Thra and Thrb. Both genes can be alternatively spliced, and nine peptide isoforms have been isolated. Three functional peptides have been identified: {alpha}1 (also known as THRA1) from Thra, and ß1 and ß2 (sometimes referred to as THRB1 and THRB2, respectively) from Thrb. The remaining six receptor isoforms lack portions of the DNA-binding and/or ligand-binding domains, and their functions remain unclear. Binding assays have shown that TR is expressed in developing testis [13, 14], and we, along with others, have reported that TR{alpha}1 mRNA and protein are abundant in the developing testis and, specifically, in Sertoli cells [15, 16]. Furthermore, both TR{alpha}2 and TR{alpha}3 (also known as Thra2 and Thra3, respectively) mRNA are expressed in Sertoli cells [15–17]. Both TR{alpha}2 and TR{alpha}3 do not mediate T3 signaling, because neither isoform binds T3 [18]. However, TR{alpha}2 binds to thyroid hormone-responsive elements in the absence of ligand, and as a result, TR{alpha}2 may be a constitutive antagonist in T3 signaling capable of partially silencing T3-mediated gene expression [19]. Therefore, T3 signaling through TR{alpha} within neonatal Sertoli cells, in previous studies as well as in the present experiments, is presumed to be through the TR{alpha}1 isoform.

A sizeable amount of literature also indicates that TRß1 could be involved in Sertoli cell development, although this area is controversial. Tagami et al. [20] reported expression of TRß1 in developing rat testes, and the concentrations of TRß1 in these testes were equivalent to the concentrations of TR{alpha}1. These findings were corroborated by a recent report showing that TRß1 mRNA is expressed in the testis of both juvenile and adult rats [21]. Neither of these studies, however, addressed the question of which cell type in the testis expressed TRß1, but other work has indicated that TRß1 mRNA is specifically expressed in the Sertoli cells of prepubertal rats [22, 23]. In addition, TRß1 mRNA is expressed in Sertoli cells of developing boar testes [22], and this initial finding, using a polymerase chain reaction methodology, has been confirmed by the recent report that TRß1 immunostaining could be detected in the developing boar testis using an antibody that specifically recognized the 55-kDa TRß1 and did not cross-react with TR{alpha}1 [3]. In contrast, others have reported that they could not detect TRß1 mRNA or protein in a series of studies involving both rat and human Sertoli cells [15, 17, 24]. Because TRß2 has a restricted distribution in the nervous system, it is unlikely to be involved in Sertoli cell development. Therefore, all T3 actions mediated through TRß within the testis are assumed to be mediated through TRß1.

These data indicate that TR{alpha}1 and/or TRß1 could potentially mediate the effects of T3 on Sertoli cells. Despite advances in our understanding of T3/TR signaling in Sertoli cells, the relative roles of TR{alpha}1 and TRß1 in Sertoli cell maturation from a mitogenic to nonmitogenic differentiating state during early postnatal life remain unresolved.

Transgenic mice lacking Thra or Thrb and, thus, all of either TR{alpha} or TRß isoforms, respectively, have been developed [25–27]. To our knowledge, testicular growth, histology, and other parameters have not been examined in either Thra or Thrb knockouts (referred to hereafter as TR{alpha}KO and TRßKO, respectively), but the fertility of both knockouts, as well as of the double-knockout males lacking both TR{alpha}1 and all TRß isoforms [28], suggests that testicular development and function must not be compromised significantly. In this report, we have used TR{alpha}KO and TRßKO mice to determine the relative roles of these receptors in mediating thyroid hormone effects on Sertoli cell and testicular development. Our results suggest that signaling through TR{alpha}1 is the normal mechanism by which T3 promotes normal Sertoli cell maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Both TR{alpha}KO and TRßKO mice on a C57BL/6 background were generated by Dr. Jacques Samarut (Laboratoire de Biologie Moléculaire et Cellulaire de l'Ecole Normal Supérieure de Lyon, Lyon, France) and obtained through Dr. Roy Weiss (University of Chicago, Chicago, IL), whereas C57BL/6 wild-type (WT) mice were bred and maintained from our mouse colony as described previously [7]. Mice were housed at 25°C with 12L:12D photoperiod and were given water and a standard rodent diet ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois and conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Determination of Adult Sertoli Cell Number, Testis Weight, and DSP

Between 115 and 120 days of age, TR{alpha}KO, TRßKO, and WT male mice (n = 7–14 per genotype) were given a lethal injection of ketamine, and one testis was excised for determination of testis weight and DSP as described previously [7]. The remaining testis was fixed by vascular perfusion using 10% neutral-buffered formalin, embedded in paraffin, sectioned (thickness, 4 µm), and stained with hematoxylin/eosin for Sertoli cell enumeration. Total Sertoli cell number per testis was determined as described previously [6].

Neonatal T3 Treatment of TRKO Mice

Litters (n = 4 per treatment per genotype) were assigned randomly to euthyroid or hyperthyroid groups. The T3 (Sigma, St. Louis, MO) was dissolved in 0.025 N sodium hydroxide, then diluted in physiological saline. To make pups hyperthyroid, T3 was administered by daily subcutaneous injections of 100 µg/kg body weight in 10 µl of vehicle [8]. On Postnatal Day 10 (day of birth = Postnatal Day 0), male pups were killed for testes excision.

Determination of Sertoli Cell Proliferation

To determine Sertoli cell proliferation in T3-treated versus untreated KO and WT mice, one testis per animal from 10-day-old WT, TR{alpha}KO, and TRßKO mice (n = 4–8 per treatment) were fixed for 1 wk in 10% neutral-buffered formalin at room temperature before dehydration and paraffin embedding. Tissues were serially sectioned (thickness, 4 µm), then deparaffinized and rehydrated. Slides were placed in boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min, then allowed to cool to room temperature to facilitate antigen unmasking [29]. Endogenous peroxidase activity was quenched by incubating sections in 0.3% H2O2 for 30 min. Sertoli cells were identified through immunodetection of Wilms tumor protein (WT1) using a rabbit polyclonal immunoglobulin (Ig) G to human WT1 (Santa Cruz Biotechnology, Santa Cruz, CA). In the testis, WT1 is a constitutively expressed transcription factor found only in Sertoli cells and, thus, functions as a cell-specific marker [30]. On an adjacent serial section, cell proliferation was detected using mouse anti-human monoclonal IgG to human Ki-67 (BD Transduction Laboratories, Lexington, KY). Binding of primary antibody was localized using a horseradish peroxidase-Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) for each species and a DAB Substrate Kit (Vector) according to the supplier's instructions. Negative-control tissue sections were processed with normal goat serum instead of primary antibody to determine nonspecific staining. Following immunostaining, sections were counterstained with Hematoxylin QS (Vector). The number of proliferating Sertoli cells was determined by Ki-67 staining, which is indicative of cell proliferation [31], in 500 Sertoli cells identified by positive WT1 staining.

Determination of Sertoli Cell Maturation Using p27Kip1, Seminiferous Tubule Patency, and Luminal Diameter as Markers

Progression through the cell cycle is regulated, in part, by the kinase activity created from complexes formed between cyclins D and E (also known as CCND1 and CCNE1, respectively) and cyclin-dependent kinase (CDK)-4, CDK6, and CDK2. The CDK inhibitors, such as p27Kip1 (also known as CDKN1B), inhibit this kinase activity and, thus, play a critical role in transition through the G1 checkpoint. Expression of p27Kip1 protein is highest in cells that have withdrawn from the cell cycle compared to cells in G1 even though they have equivalent p27Kip1 mRNA levels [32]. In both rapidly proliferating neonatal Sertoli cells [33] and Sertoli cell tumors [34], p27Kip1 expression is low. Cessation of Sertoli cell proliferation coincides with cellular maturation, and nonproliferating Sertoli cells express high levels of p27Kip1 [33, 35, 36]. Therefore, intensity of p27 expression serves as a qualitative marker of Sertoli cell maturation. To assess Sertoli cell maturation, immunostaining for p27Kip1 was performed in T3-treated and control testes from 10-day-old TR{alpha}KO, TRßKO, and WT pups (n = 4–8 per treatment) as described previously [35].

To examine Sertoli cell maturation and secretory activity further, seminiferous tubule luminal patency and diameter were evaluated. A minimum of 500 cross-sectioned seminiferous tubules were evaluated in T3-treated and control testes from 10-day-old TR{alpha}KO, TRßKO, and WT pups, and the percentage of patent lumens was calculated. Seminiferous tubule luminal diameter was measured using ImageJ software (NIH, Bethesda, MD) in a minimum of 80 cross-sectioned tubules per treatment group and genotype.

Effects of Neonatal Hypothyroidism on Sertoli Cells in TRßKO Mice

Initial results demonstrated that the effects of T3 were mediated through TR{alpha}1. To corroborate these results and determine if TRß1 plays a critical role in establishing the increased Sertoli cell population following hypothyroidism, TRßKO mice were subjected to transient neonatal hypothyroidism. The TRßKO litters (n = 2 per treatment) were randomly assigned to euthyroid and hypothyroid groups. Pups were made hypothyroid by the addition of 0.1% 6-propyl-2-thiouracil (PTU) to the dam's drinking water from birth until Postpartum Day 25 as described previously [7]. Drinking water for the euthyroid litters was untreated. On Postnatal Day 26, pups were weaned and returned to tap water to allow recovery to euthyroidism. Sertoli cell number was calculated in PTU-treated and control TRßKO mice between Postnatal Days 58 and 60 as described above. Initially, we also planned to determine the effects of transient neonatal hypothyroidism on Sertoli cells in TR{alpha}KO mice. However, this did not prove to be feasible in TR{alpha}KO mice because of impaired maternal behavior and consequent high neonatal mortality resulting from any treatment of the dam.

Statistical Analysis

All data are presented as the mean ± SEM. Comparisons of means for Sertoli cell number, testis weight, and DSP in WT, TR{alpha}KO, and TRßKO mice were analyzed using one-way ANOVA followed by the Tukey honestly significant difference (HSD) multiple-comparison procedure. Seminiferous tubule patency data were coded for open or closed lumen before performing contrast analyses [37, 38] to determine differences. Categorical variables were coded in model before seminiferous tubule luminal diameter and square root-transformed Sertoli cell proliferation data were analyzed using one-way ANOVA followed by Tukey HSD multiple comparisons. Differences in testis weights and Sertoli cell number between control and PTU-treated TRßKO mice were analyzed by Student t-test. All statistical models initially included blocking for litter. However, litter was removed from models when significance was not reached. Differences were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Loss of TR{alpha} or TRß on Adult Sertoli Cell Number, Testis Weight, and DSP

Despite a trend (P = 0.08) toward increased Sertoli cell numbers in TR{alpha}KO mice, adult Sertoli cell numbers in 120-day-old TR{alpha}KO mice (4.1 ± 0.4 x 106) did not significantly differ from those in WT controls (3.2 ± 0.4 x 106) (Fig. 1A). Although not significantly different (P = 0.26), Sertoli cell number in TRßKO mice (2.7 ± 0.2 x 106) was lower than that in WT controls. Testis weights showed significant 20% increases in TR{alpha}KO mice compared to WT mice (Fig. 1B), whereas TRßKO testes were similar to WT testes. However, body weights of TR{alpha}KO (31.7 ± 0.9 g) and TRßKO (30.7 ± 1.3 g) mice did not differ (P = 0.27) from those of WT mice (33.2 ± 1.0 g). Neither TR{alpha}KO nor TRßKO DSP values differed from WT values (Fig. 1C), although the TR{alpha}KO values again showed a trend toward an increase compared to the WT values (P = 0.07).



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  		FIG. 1. Effects of loss of TR on Sertoli cell number (A), testis weight (B), and DSP (C) in testes from 4-mo-old WT, TR{alpha}KO, and TRßKO mice. Despite a strong trend for increased Sertoli cell number and DSP, loss of TR{alpha} did not result in significant differences from WT mice. In contrast, testis weights were significantly increased in TR{alpha}KO compared to WT mice. Loss of TRß did not produce significant changes in Sertoli cell number, testis weight, or DSP compared to WT mice. Data are shown as the mean ± SEM (n = 7–14 testes/genotype). Values that do not share a common superscript are significantly different

Inhibition of Sertoli Cell Proliferation in TR{alpha}KO and TRßKO Mice Following T3 Injections

Sertoli cell proliferation, as determined by labeling index, was 3.2% in 10-day-old euthyroid WT mice, which is consistent with previously reported values [4, 7, 8]. In 10-day-old untreated WT mice, Sertoli cell proliferation was eightfold greater than that in mice treated from birth with T3 (3.2% vs. 0.4% respectively) (Fig. 2). Similarly, Sertoli cell proliferation was sixfold greater in untreated TRßKO mice compared to that in T3-treated TRßKO mice (0.6% vs. 0.1%, respectively) (Fig. 2). However, the number of Sertoli cells proliferating in 10-day-old untreated TRßKO mice was 20% of that in untreated WT mice (WT, 3.2%; TRßKO, 0.6%). Sertoli cell proliferation in untreated 10-day postnatal TR{alpha}KO mice was 3.2%, which is consistent with Sertoli cell proliferation in age-matched untreated WT mice. In contrast to WT and TRßKO mice, T3-treated TR{alpha}KO mice still exhibited significant proliferation following T3 treatment. Sertoli cell proliferation in T3-treated TR{alpha}KO mice showed a modest reduction that did not reach significance (P = 0.06) compared to that in untreated TR{alpha}KO controls, and this response was far less than that seen in WT and TRßKO mice in response to T3. This resulted in a labeling index in the T3-treated TR{alpha}KO mice (2.0%) that was far greater than that in the T3-treated WT and TRßKO mice, which had Sertoli cell proliferation of only 0.4% and 0.1%, respectively.



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  		FIG. 2. Sertoli cell proliferation in untreated and T3-treated neonatal WT, TR{alpha}KO, and TRßKO mice. Mice were treated with vehicle or T3 (100 µg/ kg body weight) from birth to Postnatal Day 10, and testes were evaluated for Sertoli cell proliferation. Sertoli cell proliferation was similar in untreated WT and TR{alpha}KO mice but significantly reduced in untreated TRßKO mice. Treatment with T3 significantly inhibited Sertoli cell proliferation in neonatal WT and TRßKO mice compared to their untreated controls of the same genotype. In contrast, T3-treated TR{alpha}KO mice did not show significantly reduced proliferation compared to untreated TR{alpha}KO mice. Data are shown as the mean ± SEM (n = 4–11 testes/ genotype/treatment). Values that do not share a common superscript are significantly different

T3 Increases Sertoli Cell p27Kip1 Expression in WT but not TR{alpha}KO Mice

Expression of p27Kip1 in Sertoli cells was compared in untreated and T3-treated, 10-day-old WT, TR{alpha}KO, and TRßKO mice. In all seminiferous tubule sections analyzed, immunodetection of p27Kip1 expression was limited to Sertoli cells, which is consistent with previous reports [33]. Sertoli cells from untreated WT mice displayed heterogeneity in nuclear p27Kip1 protein expression (Fig. 3A), with the intensity of staining ranging from light to moderately heavy. In contrast, T3-treated WT mice had consistently heavy nuclear p27Kip1 expression in all Sertoli cells (Fig. 3B). Untreated TR{alpha}KO mice also showed heterogeneity in Sertoli cell p27Kip1 expression, with a range of staining intensity similar to that in untreated WT mice (Fig. 3C). Although most nuclear staining of p27Kip1 in untreated TR{alpha}KO Sertoli cells was light to moderately heavy, numerous Sertoli cells had minimal p27Kip1 expression. Significantly, T3 did not increase p27Kip1 expression in TR{alpha}KO Sertoli cells (Fig. 3D) compared to that in untreated TR{alpha}KO mice, which is in contrast to the marked increases seen in T3-treated WT Sertoli cells compared to those in untreated WT Sertoli cells. Instead, p27Kip1 expression in T3-treated TR{alpha}KO Sertoli cells remained heterogeneous and was similar to that in both untreated WT and TR{alpha}KO mice. Because of the already low percentage of Sertoli cells proliferating in 10-day-old untreated TRßKO mice, p27Kip1 expression was heavy (data not shown). Therefore, comparison of p27Kip1 expression in untreated and T3-treated TRßKO mice was not performed.



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  		FIG. 3. Effects of T3 on expression of p27Kip1 in Sertoli cells from 10-day-old WT and TR{alpha}KO mice. Either T3 or vehicle was given to neonatal mice as described in Figure 2. Immunohistochemical detection of p27Kip1 protein is indicated by brown coloration, and samples were counterstained with hematoxylin (blue). All samples were stained under identical conditions in the same staining run to allow direct comparisons. A) Sertoli cells of euthyroid WT mice showed heterogeneous nuclear staining for p27Kip1, with some cells showing more intense brown coloration (black arrow) indicative of greater maturation and withdrawal from the cell cycle. Other Sertoli cells showed minimal p27Kip1 staining (white arrow), indicating that they remain in the cell cycle. Note that only Sertoli cells stain for p27Kip1, with all germ cells showing only blue counterstain. B) Sertoli cells of T3-treated WT mice had heavily concentrated, dark-brown nuclear staining (arrowheads) indicative of high p27Kip1 protein levels and cell-cycle arrest. C and D) Sertoli cells of euthyroid (C) and T3-treated (D) TR{alpha}KO mice displayed a pattern of heterogeneous expression of p27Kip1 (black and white arrows), indicating that T3 did not alter p27Kip1 expression and that T3 effects on Sertoli cell maturation are mediated through TR{alpha}1. Bars = 25 µm

T3 Does Not Increase Seminiferous Tubule Patency or Luminal Diameter in TR{alpha}KO mice

During the early postnatal period, when Sertoli cell proliferation declines and then finally ceases, the solid seminiferous cords have not yet canalized to form the seminiferous tubules. Significant seminiferous tubule canalization and lumen formation normally begins during the latter part of the second postnatal week of life in rodents as a result of seminiferous fluid production by maturing Sertoli cells [8, 39]. Therefore, luminal patency and diameter are indicators of the onset and magnitude, respectively, of adluminal fluid production by maturing Sertoli cells. Consistent with these previous results, the percentages of seminiferous tubules with patent lumens on Postnatal Day 10 were similarly low in untreated WT, TR{alpha}KO, and TRßKO mice (Figs. 4 and 5A). However, T3 treatment starting at birth resulted in 19- and 12-fold increases in tubule patency in 10-day-old WT and TRßKO mice, respectively, compared to that in their untreated controls. In contrast, seminiferous tubules in TR{alpha}KO mice did not show increased luminal canalization in response to T3.



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  		FIG. 4. Ability of T3 treatment to induce precocious seminiferous tubule canalization in 10-day-old WT, TR{alpha}KO, and TRßKO mice. Either T3 or vehicle was given to neonatal mice as described in Figure 2. Seminiferous tubules in WT controls (A) showed minimal canalization on Postnatal Day 10. However, T3 treatment of WT mice (B) resulted in significant increases in the number of patent tubules. Although seminiferous tubule canalization in control TR{alpha}KO mice (C) was similar to that in control WT mice, loss of TR{alpha} precluded seminiferous tubule canalization induced by T3 treatment (D). Canalization of seminiferous tubules in control (E) or T3-treated (F) TRßKO mice resembled that in the control and T3-treated WT mice, respectively, indicating that the T3 effects on canalization did not require TRß. Bars = 50 µm

Luminal diameters of seminiferous tubules in 10-day-old untreated TRßKO mice were comparable to those in control WT mice, whereas luminal diameters in TR{alpha}KO mice were smaller than those in WT controls (Fig. 5B). As expected, T3 increased seminiferous tubule luminal diameter by sevenfold in WT testes compared to those from untreated WT mice. Likewise, TRßKO mice exhibited eightfold increases in tubule diameter after T3 treatment compared to control TRßKO testes (Figs. 4 and 5B). In striking contrast to WT and TRßKO mice, mean luminal diameter in TR{alpha}KO mice was not significantly increased by T3 treatment. This resulted in luminal diameters of patent seminiferous tubules in T3-treated TR{alpha}KO mice that were 90% smaller than the patent tubules in either T3-treated WT or TRßKO tubules.



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  		FIG. 5. Ability of thyroid hormone to induce maturation and adluminal secretions in Sertoli cells from WT, TR{alpha}KO, and TRßKO mice. A) Canalization of seminiferous tubules in 10-day-old WT, TR{alpha}KO, and TRßKO mice treated with vehicle or T3. Treatment with T3 induced precocious canalization of seminiferous tubules in WT and TRßKO mice but did not affect TR{alpha}KO mice. B) Effects of T3 on seminiferous tubule luminal diameter in WT, TR{alpha}KO, and TRßKO mice. In WT and TRßKO mice, T3 increases seminiferous tubule luminal diameter, an indicator of adluminal fluid secretion of maturing Sertoli cells. In contrast, TR{alpha}KO mice do not show an increased seminiferous tubule luminal diameter in response to T3. Data are shown as the mean ± SEM (n = 4–8 testes/genotype/treatment). Values that do not share a common superscript are significantly different

TRßKO Mice Respond Normally to Transient Neonatal Hypothyroidism

Consistent with previous reports [7], PTU treatment increased testis weights (data not shown) and Sertoli cell number (Fig. 6) in 60-day-old WT mice. After recovery from transient neonatal hypothyroidism, testis weights in 60-day-old TRßKO mice (111 ± 2 mg) were 38% greater than those in untreated TRßKO controls (80 ± 2 mg). Similarly, PTU treatment resulted in a significant 31% increase in adult Sertoli cells in TRßKO mice following transient neonatal hypothyroidism (3.8 ± 0.3 x 106) compared to that in untreated TRßKO mice (2.9 ± 0.2 x 106) (Fig. 6). Critically, the increase in Sertoli cell number in TRßKO mice after PTU treatment was similar to that observed in PTU-treated WT mice (Fig. 6).



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  		FIG. 6. Effect of transient neonatal hypothyroidism on testis weight and Sertoli cell number in TRßKO mice. At 60 days of age, Sertoli cell number was calculated in WT and TRßKO mice that were either untreated or treated neonatally with PTU. Transient neonatal hypothyroidism resulted in comparable increases in Sertoli cell number in WT and TRßKO mice. Data are shown as the mean ± SEM (n = 6–8 testes/treatment). Asterisks denote significant differences from untreated control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Expression of TR in rat and human testes is maximal during late fetal and early neonatal life, when Sertoli cell proliferation is high; TR levels subsequently decline as Sertoli cell proliferation ceases [13, 17, 23, 30]. In rats, serum thyroid hormone concentrations rise markedly during early postnatal life [40], a time when expression of both TR{alpha}1 and TRß1 in Sertoli cells has been demonstrated [3, 13, 15, 17, 23, 24, 41, 42], suggesting that either or both may play a role in regulation of Sertoli cell proliferation. In the present study, we used knockout mice lacking either {alpha} or ß isoforms of TR to determine if the inhibition of proliferation and stimulation of maturation in Sertoli cells by T3 occurs through TR{alpha}1 and/or TRß1.

Our results show that TR{alpha}1, rather then TRß1, is critical for T3 actions on Sertoli cell development in mice. Luminal canalization in seminiferous cords, which begins during the second week of postnatal life and is stimulated by T3, is a useful indicator of T3 effects on Sertoli cells [2, 8, 39, 43]. Seminiferous tubule patency was relatively low in untreated mice of all genotypes at 10 days of age but was significantly greater in TRßKO than in TR{alpha}KO mice, suggesting that TR{alpha}1 was critical for luminal canalization. Hyperthyroidism promoted Sertoli cell maturation and seminiferous tubule canalization in WT mice, as shown by marked increases in luminal patency and diameter, respectively, compared to euthyroid controls, which is consistent with previous findings [8]. Treatment with T3 resulted in a similar response in TRßKO mice, with comparable increases in luminal patency and diameter. In contrast, T3 treatment was unable to increase seminiferous tubule canalization or diameter in TR{alpha}KO mice. The inability of T3 to induce seminiferous tubule luminal opening in TR{alpha}KO mice, but not in TRßKO mice, illustrates that TR{alpha}1 is necessary for mediating the primary effects of T3 on Sertoli cell maturation.

Despite their overall initial decreases in Sertoli cell proliferation, TRßKO mice showed further reductions in Sertoli cell proliferation following neonatal hyperthyroidism, indicating that the absence of TRß1 does not preclude inhibitory T3 effects on Sertoli cell proliferation. In contrast to that in both WT and TRßKO mice, Sertoli cell proliferation in TR{alpha}KO mice was not significantly reduced following T3 treatment. In addition, p27Kip1 expression in T3-treated TR{alpha}KO mice remained light to moderately heavy in intensity, similar to that in untreated WT and TR{alpha}KO mice and contrasting with the heavy p27Kip1 nuclear staining in Sertoli cells from T3-treated WT mice, suggesting that TR{alpha}1 is necessary for the inhibition of Sertoli cell proliferation and maturation by T3. Moreover, if TRß1 was the primary mediator of the T3 effect on Sertoli cell proliferation, then TR{alpha}KO mice should have shown a hyperresponsivity to T3 treatment because of the loss of constitutive silencing by TR{alpha}2 [19].

The trend toward a diminished Sertoli cell response in T3-treated TR{alpha}KO mice may be more reflective of secondary endocrine effects of hyperthyroidism than that of direct effects on Sertoli cells. Recent evidence has shown that hyperthyroidism decreases FSH and insulin-like growth factor (IGF)-I in males [44]. Both FSH and IGF-I increase proliferation in neonatal Sertoli cells [45, 46], a time when FSH-receptor expression normally is rising [47], and knockout mice lacking either FSH or IGF-I have decreased numbers of Sertoli cells and smaller testes [48–50], illustrating that these hormones are critical mitogens in neonatal Sertoli cells. Therefore, a reduction in FSH and/or IGF-I as a result of T3 treatment may be involved in the modest decrease in Sertoli cell proliferation in TR{alpha}KO mice even in the absence of direct T3 effects on Sertoli cell proliferation.

The inability of T3 to produce precocious luminal canalization in the absence of TR{alpha}1 also is consistent with the hypothesis that reduced Sertoli cell proliferation in T3-treated TR{alpha}KO mice results from secondary effects, such as reduced FSH and IGF-I, rather than from signaling through TRß1. Taken together, the luminal canalization and Sertoli cell proliferation data support the hypothesis that TR{alpha}1 plays the critical role in neonatal Sertoli cell proliferation and maturation.

The TRß1 is important for thyroid hormone negative feedback, and TRßKO mice have elevated TSH and mild hyperthyroidism [25]. Therefore, the reduction in Sertoli cell proliferation in 10-day-old untreated TRßKO (0.6%) compared to WT (3.2%) mice may reflect exposure to elevated T3 levels during neonatal life. This hypothesis is supported by the comparable Sertoli cell proliferation observed between TRßKO and WT mice at Postnatal Day 5 (data not shown), which became significantly reduced in TRßKO compared to WT mice by 10 days. Therefore, the fact that adult TRßKO and WT Sertoli cell populations do not differ at 120 days of age may be a result of the similar percentages of proliferating Sertoli cells at Postnatal Day 5, when a higher percentage of Sertoli cells are in the cell cycle. However, other possibilities not explored in the present study include an extended period of low Sertoli cell proliferation or a reduction in apoptosis [51–53] in TRßKO compared to WT mice. Moreover, the decreased proliferation of TRßKO Sertoli cells, most likely in response to increased endogenous thyroid hormone, again suggests that TRß1 is not the receptor responsible for inhibitory effects of T3 on Sertoli cell proliferation. This is consistent with the conclusion from data indicating that T3 treatment of TRßKO mice produced a further reduction in Sertoli cell proliferation that was qualitatively similar to that in T3-treated WT mice.

The conclusion that TR{alpha}1 mediates T3 effects on Sertoli cells obtained from the hyperthyroidism experiments can be examined directly by determining whether hypothyroidism can increase Sertoli cell proliferation normally in the absence of TRß1. Our data indicate that TRß1 is not necessary for increases in Sertoli cell populations after transient neonatal hypothyroidism, based on similar increases in Sertoli cell number and testis weight in adult WT and TRßKO mice following neonatal PTU treatment. These data emphasize that hypothyroidism effects on Sertoli cells occur normally in TRßKO mice and that T3 regulates Sertoli cell proliferation through TR{alpha}1 rather than TRß1. Finally, treating TR{alpha}KO litters with PTU could show definitively that the presence of TRß1 is not sufficient to allow increases in Sertoli cell number induced by neonatal hypothyroidism. However, this complementary experiment could not be conducted because of difficulties in establishing pregnancies and high neonatal mortality in these mice, which was exacerbated when pups and, especially, dams were subjected to any treatment.

If TR{alpha}1 is necessary for T3 effects on Sertoli cells, then loss of TR{alpha}1 in the TR{alpha}KO mice should induce phenotypic changes comparable to those seen with hypothyroidism. Sertoli cell number, DSP, and subsequent testis weights show increases of approximately 40% following recovery from transient neonatal hypothyroidism [7]. Similarly, TR{alpha}KO mice had 10–30% increases in adult Sertoli cell number, testis weight, and DSP compared to WT mice, although some of these changes did not achieve statistical significance. Critically, the increases in TR{alpha}KO testis weights were not allometric, because body weights were similar to those of WT controls. Although the TR{alpha}KO mice do not totally recapitulate the effects of hypothyroidism on Sertoli cell number, testis weight, or DSP, a strong trend exists toward increases in these parameters that also are increased by neonatal hypothyroidism. These findings are consistent with, and further confirm, the idea of TR{alpha}1 as the predominate receptor isoform for T3 effects in neonatal Sertoli cells.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Jacques Samarut (Laboratoire de Biologie Moléculaire et Cellulaire de l'Ecole Normale Supérieure de Lyon, Lyon, France) and Dr. Roy Weiss (University of Chicago, Chicago, IL) for providing the transgenic mice used in the present study, Dr. David Schaeffer for help with performing statistical analyses, Angela Oki for help with immunostaining, and Melissa Zakroczymski for assistance with figure preparation and manuscript submission.


   FOOTNOTES
 
1 Supported by NIH grant ES11590 (to P.S.C.), a grant from the Lalor Foundation (to D.R.H.), and the Thanis A. Field Endowment. D.R.H. also was supported by postdoctoral fellowships from the Lalor Foundation and Reproductive Biology Research Training Program (NIH grant T32 HD07028), University of Illinois-Urbana. The present investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR16515 from the National Center for Research Resources, National Institutes of Health. Back

2 Correspondence: Paul S. Cooke, Department of Veterinary Biosciences, 2001 S. Lincoln Ave., University of Illinois at Urbana-Champaign, Urbana, IL 61802. FAX: 217 244 1652; p-cooke{at}uiuc.edu Back

Received: 1 March 2005.

First decision: 25 March 2005.

Accepted: 25 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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