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Department of Human Genetics, University of Michigan Medical School (J.H.S., S.K.K., M.L.B., T.L.G., D.E.W.-C., S.A.C.), Ann Arbor, Michigan 48109; the Department of Human Genetics, Mount Sinai School of Medicine (A.C.-B.), New York, New York 10029-6514; and the Department of Laboratory Medicine and Pathology, Mayo Clinic (R.V.L.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. Sally Camper, 4301 MSRB III, 1500 West Medical Center Drive, Department Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638. E-mail: scamper{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit essential for TSH,
LH, and FSH activity (Gsu-/-) exhibit
hypothyroidism and hypogonadism similar to that observed in TSH
receptor-deficient hypothyroid mice (hyt) and
GnRH-deficient hypogonadal mutants (hpg). Although the
five major hormone-producing cells of the anterior pituitary are
present in Gsu-/- mice, the relative
proportions of each cell type are altered dramatically. Thyrotropes
exhibit hypertrophy and hyperplasia, and somatotropes and lactotropes
are underrepresented. The size and number of gonadotropes in
Gsu mutants are not remarkable in contrast to the
hypertrophy characteristic of gonadectomized animals. The reduction in
lactotropes is more severe in Gsu mutants (13-fold
relative to wild-type) than in hyt or hpg
mutants (4.5- and 1.5-fold, respectively). In addition, T4
replacement therapy of Gsu mutants restores
lactotropes to near-normal levels, illustrating the importance of
T4, but not -subunit, for lactotrope proliferation and
function. T4 replacement is permissive for gonadotrope
hypertrophy in Gsu mutants, consistent with the role
for T4 in the function of gonadotropes. This study reveals
the importance of thyroid hormone in developing the appropriate
proportions of anterior pituitary cell types. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and unique ß-subunits that confer
specificity and bioactivity. Two functions of TSH are stimulation of
the thyroid gland and thyroid folliculogenesis in the fetus. Likewise,
LH and FSH play an integral role in gonadal differentiation in neonates
(1) and are also regulators of sperm and ovarian development (2, 3).
The gonadotropins and TSH are under feedback control by gonadal
steroids and thyroid hormone, respectively. Feedback occurs both at the
level of the hypothalamus and the pituitary gland.
Targeted disruption of the glycoprotein hormone -subunit
(Gsu or Cga) gene through homologous
recombination in mouse embryonic stem cells was used to analyze the
role of -subunit, TSH, LH, and FSH in vivo (1).
Gsu+/- mice are normal and fertile, whereas
Gsu-/- mice exhibit hypothyroidism,
hypogonadism, infertility, and severe growth deficiency. Although the
Gsu-/- mice have a normal lobular pituitary
structure, there is a profound reduction in number of somatotropes and
lactotropes as well as evidence of hyperplasia and hypertrophy of
thyrotropes. Although the thyrotrope hyperplasia was expected, the near
absence of lactotropes was not. Hypothyroidism usually leads to
elevation of TRH, which results in stimulation of PRL production (4, 5). One plausible explanation is that lactotrope differentiation is
impaired due to the lack of secreted -subunit monomers. This
hypothesis is based on the observation that -subunit stimulates PRL
production in fetal pituitary cell cultures (6). Alternatively, reduced
PRL and GH production in response to the lack of steroid hormones and
thyroid hormone, respectively, could be responsible. Finally, if
thyrotropes, somatotropes, and lactotropes are derived from a common
precursor, the hyperplasia of thyrotropes may deplete the precursor
pool available for differentiation of the other cell types.
There are two other animal models that exhibit aspects of the
Gsu-/- phenotype. The arrested thyroid
development of Gsu-/- mice is comparable to
the hypothyroid mutants (hyt), which lack a functional TSH
receptor (7). The hyt mice also exhibit hyperplasia and
hypertrophy of thyrotropes with a decrease in the population of
somatotropes (1, 8). The hypogonadism and infertility of
Gsu-/- mice are reflective of the
hypogonadal mutant (hpg), which has a deletion in the GnRH
gene, effectively eliminating hypothalamic stimulation of pituitary
gonadotropes (9). Comparison of the pituitary cellular makeup of these
mutants and that of Gsu-/- mice supports
the importance of thyroid hormone for lactotrope differentiation.
T4 replacement demonstrates that -subunit is not
required for PRL cell differentiation and that T4 is
critical for gonadotrope responsiveness to steroid hormone feedback
control.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gsu-/- mutants are embryonic stem
cell-derived mice with a targeted mutation in the CG gene
(Cgatm1). These mice were generated and
maintained at the University of Michigan and have been deposited in the
Induced Mutant Resource at The Jackson Laboratory (Bar
Harbor, ME) (1). The hyt and hpg mice were
provided by The Jackson Laboratory. All procedures using mice were
approved by the University of Michigan Committee on Use and Care of
Animals. All experiments were conducted in accordance with the
principles and procedures outlined in the NIH Guidelines for the Care
and Use of Experimental Animals.
PCR screening of Gsu-/- mutants
Gsu-/- mutants were detected by PCR
analysis of genomic DNA prepared from tail biopsies as previously
described (1). Two sets of primers were used, one to amplify the
Gsu-/- allele and another to amplify the
wild-type allele.
TSH RIA
TSH was measured by a mouse specific double antibody RIA using a
mouse TSH/LH reference preparation (AFP51718mp), a mouse TSH antiserum
(AFP98991), and rat TSH antigen for radioiodination (NIDDK rTSH-I-9).
All reagents were obtained from Dr. A. F. Parlow (Harbor
University of California-Los Angeles Medical Center, Torrance, CA). For
each serum sample, duplicate determinations were made in parallel on
25-µl aliquots of serum. The limit of sensitivity was 0.6 ng, and the
intraassay variation less than 6% (10, 11). Equivalent amounts of
hyperthyroid mouse serum were added to the standard tubes. Enzymatic
iodination of the NIDDK rTSH-I-9 preparation was achieved by the
glucose oxidase/lactoperoxidase technique (12). After labeling
[125I]rTSH was purified by concanavalin A-Sepharose
adsorption and stored in aliquots at -80 C. The tracer was further
purified by high resolution gel filtration with Sephadex G-100
performed immediately before use.
Thyroid hormone treatment
Two approaches to thyroid replacement therapy were taken. First,
adult Gsu-/- mice at 56 days (8 weeks) of
age were injected daily with 2.0 µg T4 (Sigma T-0397) for
40 days. This regimen is similar to one previously described (13, 14).
Second, neonates were fed a special chow supplemented with powdered
thyroid glands (250 mg thyroid powder/kg chow) for 8 weeks. The thyroid
chow was obtained from Amersham (Arlington Heights, IL).
All mice were weighed on an Ohaus GA110 scale once a week
(Ohaus, Union, NJ). Measurements were segregated according to
phenotype, sex, and type of treatment. The average and SD
of the mean were calculated for each group. The unpaired t
test was applied to weight data collected from normal mice (+/- and
+/+; n = 8, 4 females and 4 males), untreated -/- mice (n =
7; 2 females and 5 males), -/- mice injected with T4
(n = 7; 3 females and 4 males), and -/- mice fed thyroid chow
(n = 8; 3 females and 5 males) and their normal littermates fed
the same diet (+/+ and +/-; n = 21; 10 females and 11 males).
Mice were killed at the end of treatment, and pituitaries of all the
mice were removed and analyzed by immunohistochemistry. Seven
additional male -/- mice (4 months) were included in the
immunohistochemical analysis to enhance the statistical significance of
the quantitation (15).
Injection of 4-month-old mice with 2.0 µg T4 (Sigma T-0397) was also performed. Injections occurred once a day for 3 days, after which the mice were killed, and pituitaries were removed and analyzed (n = 4).
Pituitary histology and immunohistochemistry
Pituitaries were removed and fixed in 4% paraformaldehyde, pH
7.2, for 3–4 h and embedded in paraffin 3-µm sections and
immunostained with antibodies to five of the pituitary hormones
followed by counterstaining with hematoxylin. Gill’s formulation 3
hematoxylin was obtained from Fisher Scientific
(Pittsburgh, PA). Pituitaries were immunostained with polyclonal
antiserum against rat GH (1:1000 dilution), rat LHß (1:2000), rat
TSHß (1:1000), rat PRL (1:2000), and human ACTH (1:1000) and
counterstained with hematoxylin. All antisera except ACTH antiserum
were donated by the National Hormone and Pituitary Program, NIDDK
(Bethesda, MD). ACTH antiserum was obtained from DAKO Corp. (Santa Barbara, CA). Biotinylated secondary antibodies
were used in conjunction with avidin and biotinylated peroxidase
(Vectastain guinea pig, rabbit, human kits, Burlingame, CA).
Diaminobenzidine was used as the chromogen resulting in brown
staining.
The percentages of the GH, LH, and PRL cell types in the pituitary were counted under x40 brightfield illumination with a reticular eyepiece (1-mm2 grid area). The total number of cells and the positively stained cells were counted separately within each grid area. At least 1500 cells were counted and at least 3 different fields were examined in each animal. The numbers from multiple grid counts were tallied, and the percentage of positively stained cells was calculated. The following sets of mice were quantitated: 2 females and 1 male, +/+; 1 female and 8 males, untreated -/-; 2 females and 3 males, -/- injected with T4; and 4 females and 3 males, -/- raised on thyroid chow.
Morphometric analysis
The diameter of 50 thyrotropes was measured in photographs of
pituitary sections from +/+ and -/- mice. For each thyrotrope, two
measurements were taken at the widest part of the cell at 90° angles
to one another. The average diameter (12.4 ± 2.8 µm for -/-
and 6.6 ± 1.3 µm for +/+) was used to calculate the area
(A = 
r2) and volume (V = 4/3
r3) of each cell.
RNA analysis
Pituitaries from Gsu-/- and
Gsu+/+ mice were collected and pooled
according to genotype. RNA was purified using the either the Trizol
prep (Life Technologies, Grand Island, NY) or the RNAzol
prep (16). Three to 5 µg RNA were loaded into each lane of a 1.5%
agarose gel and electrophoresed in 1 x
3-[N-morpholino]propanesulfonic acid buffer at 80 mA for
6–10 h. The RNA was transferred overnight onto a Zeta-Probe Blotting
Membrane (Bio-Rad Laboratories, Inc., Hercules, CA).
Filters were hybridized with [-32P]deoxy-CTP-labeled
mouse TSHß, LHß, PRL, GH, rat POMC, FSHß, and mouse ß-actin
(see below) at 65 C overnight, then washed to a stringency of 0.1
x SSC (standard saline citrate)-0.1% SDS at 57–65 C (17). Filters
were exposed on film for up to 2 weeks or to PhosphorImager screens
overnight (Molecular Dynamics, Sunnyvale, CA). Quantitation was carried
out with a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). This
experiment was repeated with independent RNA preparations. Filters were
stripped by soaking in boiled 1% SDS-0.1 x SSC and 40
mM Tris-Cl, pH 7.5. Blots were normalized assuming that the
ß-actin hybridization signal from +/+ and -/- pituitary RNA samples
should be the same.
The complementary DNA (cDNA) probes used for Northern analysis were provided by Richard Maurer (FSHß, rPRL), David Gordon and E. Chester Ridgway (mouse TSHß), John Nilson (mouse LHß), and Michael J. Dixon (rat POMC). The mouse GH probe consists of 802 bp spanning from 59 bp upstream of the ATG to 41 bp downstream of the stop codon. The rat PRL probe is a 735-bp cDNA fragment including most of the 5'-untranslated region, the complete coding region, and a portion of the 3'-untranslated region. The 874-bp rat FSHß cDNA probe contains the complete coding sequence. The POMC probe is a 900-bp cDNA fragment isolated by PCR amplification. The TSHß probe is a 496-bp HindII-EcoRI fragment of the mTSHß cDNA cloned into pGEM3. The LHß probe was generated from a 250-bp fragment of the mouse LHß cDNA isolated by PCR amplification and cloned into pBSK-. Mouse ß-actin is from CLONTECH Laboratories, Inc. (Palo Alto, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gsu-/- mice exhibit
thyrotrope hypertrophy and hyperplasia Gsu-/- mice are devoid of -subunit
messenger RNA (mRNA) and protein (1). Heterodimer formation between
TSHß and -subunit is essential for ß-subunit secretion (18, 19, 20, 21).
Therefore, it was not expected that TSHß could be secreted in the
absence of -subunit. No TSH was detectable in the serum of
Gsu-/- mice by RIA (<20 ng/ml, n = 3
-/- mice; 86.4 ± 2.9 ng/ml, n = 3 +/+ mice). In addition,
no secretory granules were evident in thyrotropes of -/- mice
examined by electron microscopy (data not shown). T4 was
also undetectable in the serum of Gsu-/-
mice by RIA, consistent with the lack of TSH stimulation (1).
The cellular composition of Gsu-/- mouse
pituitaries is distorted relative to that of normal mice (1).
Thyrotrope hypertrophy and extensive, diffuse hyperplasia were present
in 2-month-old mutants (Fig. 1
).
Morphometric analysis revealed that the average diameter of the
thyrotropes in -/- mice was increased approximately 1.9-fold relative
to that of +/+ mice (12.4 ± 2.8 and 6.68 ± 1.3 µm,
respectively), resulting in a 6.8-fold increase in cell volume. Despite
this increase in volume and the dramatic hyperplasia of thyrotropes,
pituitaries of Gsu-/- mice weigh
approximately the same as those of normal mice at 2 months of age (2 mg
average; data not shown). Therefore, the increase in thyrotrope size
and number must occur at the expense of other cell types. Both GH- and
PRL-producing cells appear to be profoundly reduced in number by
immunohistochemistry (Figs. 1 and 2).
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). In
wild-type pituitaries, an average of 38% and 49% of the cells
immunostained with PRL and GH antibodies, respectively. In
Gsu-/- mice, lactotropes and somatotropes
averaged only 3% and 12.5% of the cells, representing 13- and 4-fold
reductions, respectively. The modest increase in gonadotropes in
Gsu-/- mice relative to control values was
not statistically significant (7% vs. 5.2%,
respectively).
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Gsu-/- mice were reduced to 0.27% of those
in normal mice (Fig. 4). GH mRNA levels
in -/- pituitaries were only 7.9% of the normal levels. The FSHß
and LHß mRNA levels were similar to those in +/+ mice (174% and
106%, respectively), and POMC mRNA levels were slightly reduced
(mutants were 39% of normal).
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Gsu-/- proportionate dwarfism Gsu-/- mutants (n = 7) were injected
with T4 every day for 40 days in an attempt to reverse the
established phenotype (13, 14). Both treatments relieved the
proportionate dwarfism characteristic of
Gsu-/- mutants, indicating that both
regimens are effective (Fig. 5 and Table 1). The catch-up growth stimulated by
thyroid hormone treatment of 2-month-old mice resulted in an 86%
weight gain over the 40-day treatment period compared with the modest
gain of untreated mutants (16–18%). After treatment, the weights of
male and female Gsu-/- mice were equivalent
to those of female control mice. Male control mice were heavier than
female controls and Gsu-/- mice of both
sexes as expected due to the hypogonadism of the
Gsu-/- mice (1). The size and weight of
Gsu-/- neonates fed thyroid hormone in
their chow was indistinguishable from those of their normal female
littermates at 8 weeks (P > 0.1) and slightly less
than those of their male littermates (P < 0.05; Table 1).
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Gsu-/-
mice showed reduced production of TSHß by immunohistochemistry (Fig. 1
). T4-treated adults contained TSHß-staining cells that
appeared shrunken relative to those of untreated adult
Gsu-/- mice. Treatment with T4
from birth profoundly reduced the number of TSHß-staining cells.
Thyrotropes were rare in these mice relative to those in normal mice.
Both thyroid hormone replacement regimens resulted in striking
gonadotrope hypertrophy, as illustrated by the LHß antibody staining,
and each treatment increased gonadotrope cell number significantly
relative to untreated Gsu-/- mice (Fig. 3).
The GH and PRL immunostaining suggested that both T4
treatments substantially increased the somatotrope and lactotrope cell
numbers in Gsu-/- mice (Figs. 1 and 2), and
the cell counts corroborated this (Fig. 2). Somatotrope cell numbers in
T4-treated Gsu-/- mice were
indistinguishable from normal values. Lactotrope numbers were
dramatically improved (23% and 26% in treated adults and neonates),
but they remained slightly less than normal (38%).
The increase in PRL and GH cells in Gsu-/-
mice after 40 days of T4 treatment could be attributable to
differentiation from precursor cells. Alternatively, T4
treatment may have enhanced hormone production or storage in
differentiated cells already present but inactive. To distinguish
between these possibilities, a 3 day T4 treatment was
performed. Four Gsu-/- mice were injected
with T4. Immunostaining for TSH, PRL, GH, and LH revealed
no difference between the untreated mice and the 3-day treated mice
(data not shown). Thyrotropes remained hypertrophic and hyperplastic.
PRL cell counts were similar in the 3-day treated
Gsu-/- mutants (2.9%) and untreated
Gsu-/- mutants mice (3%). This result
suggests that few inactive lactotropes are present.
Lactotrope content is reduced more in hypothyroid mice than in
hypogonadal mice
The pituitaries of Gsu-/- mice
were compared with TSH receptor-deficient hypothyroid (hyt)
and GnRH-deficient hypogonadal (hpg) mutants. Hypothyroid
hyt/hyt mice had fewer somatotropes than normal mice (33%
vs. 49%), but more than untreated
Gsu-/- mutants (12.5%). Both the
hypothyroid hyt/hyt and the hpg/hpg mice have
fewer lactotropes (7% and 25%, respectively) than normal mice (38%).
Neither the hpg nor the hyt mutants had as severe
a reduction in lactotropes as the Gsu-/-
mice (3%). This suggests that both thyroid hormone and steroid
hormones are important for normal lactotrope numbers and that the
requirement for thyroid hormone is stronger than the requirement for
gonadal steroids.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit gene has a
dramatic impact on pituitary cellular composition-subunit gene caused a lack of
gonadotropins and TSH, which resulted in hypogonadism, hypothyroidism,
and proportionate dwarfism (1). The cellular composition of the
anterior pituitary was altered dramatically. Although lactotropes
normally represent 38% of the total cells, very few PRL-producing
cells were detectable by immunohistochemistry in the knockout mice
(3%). In addition to reduced PRL protein, PRL mRNA was reduced to
0.27% of normal levels. Somatotropes, another of the most abundant
cell types in normal pituitaries (49%), were reduced to 12.5% of the
total, and GH mRNA was reduced as well (7.9% of normal). These data
suggest that the targeted mutation affected PRL and GH gene
transcription and protein accumulation. The predominant pituitary cell
type in Gsu mutants was the thyrotrope. These cells
displayed both dramatic hypertrophy (
7-fold increase in cell volume)
and diffuse hyperplasia throughout the pituitary gland. Thyroid hormone
deficiency induced by thyrotoxic drugs also induces hypertrophy and
hyperplasia, but the hyperplasia is limited to discrete foci within the
gland (1, 8). The proportion of gonadotropes and corticotropes was not
substantially changed, and the levels of LH, FSH, and POMC mRNA were
similar (within 2- to 3-fold) in mutants and wild-type mice.
There were several plausible explanations for the expansion of
thyrotropes and the reduction in both somatotropes and lactotropes.
First, congenital hypothyroidism impacts thyroid development during
gestation (1, 7), potentially stimulating expansion of thyrotropes
early enough to cause pituitary-wide hyperplasia. In contrast,
hypothyroidism induced in older mice may only induce focal hyperplasia
because of a reduction in the proliferative capacity of thyrotropes
with age. Several lines of evidence support the derivation of
thyrotropes, lactotropes, and somatotropes from a common precursor cell
(22); thus, it is possible that the massive recruitment of thyrotropes
during gestation reduces the precursor pool available for
differentiation into the other two cell types. A second possibility is
based on the fact that PRL production and lactotrope proliferation are
stimulated by estrogen, and GH production is stimulated by thyroid
hormone (23, 24). Thus, the hypogonadism and hypothyroidism and the
resulting lack of estrogen and thyroid hormone could have caused the
reduction in lactotropes and somatotropes, respectively. Third, several
laboratories have demonstrated that -subunit stimulates lactotrope
differentiation (6, 25), opening the possibility that the dramatic
impact on lactotropes was caused by the disruption of the -subunit
gene. To assess the potential contribution of each of these factors to
pituitary cell content, we carried out thyroid hormone replacement and
compared the pituitary cell content of the Gsu mutants to
mice with either congenital hypogonadism or hypothyroidism.
Thyroid hormone replacement normalizes pituitary cellular
makeup
Thyroid hormone replacement rescued the proportionate dwarfism,
prevented and reversed thyrotrope hypertrophy and hyperplasia, and
normalized the number of lactotropes and somatotropes. Administration
of thyroid hormone by ip injection of young adult mice for 40 days
partially reversed the thyrotrope hypertrophy and hyperplasia. Newborns
whose mothers were fed thyroid hormone-supplemented chow and who were
fed the same chow after weaning had normalized somatotropes and
lactotropes and very few thyrotropes at 40 days of age. However, a
3-day thyroid hormone replacement regimen was not effective in inducing
PRL production. If thyroid hormone were able to induce existing
lactotrope cells in the Gsu-/- mice to
synthesize PRL mRNA and protein, then the 3-day T4
replacement regimen would have been expected to be more effective.
Transgene ablation studies have shown that renewal of depleted
lactotropes from precursor pools in adult mice requires 3–6 weeks
(26). Thus, the observed establishment of PRL production in 40 days is
consistent with significant differentiation of lactotropes from
precursor cells. The fact that thyroid hormone alone was effective in
normalizing pituitary cellular makeup suggests that somatotropes and
lactotropes require T4 for differentiation.
Thyroid hormone is important for lactotrope differentiation
Comparison of pituitary cell content in hypothyroid,
hyt, hypogonadal, and Gsu-/-
mutants supports the reliance of PRL and GH cells on T4.
The hyt/hyt mutants exhibit hypertrophy and hyperplasia of
thyrotropes and a decrease in the number of somatotropes (8). Using
immunohistochemistry, we have shown that hyt/hyt mutants
also have a profound reduction in the number of lactotropes (7% of the
cells are lactotropes), supporting the critical role of thyroid hormone
in lactotrope differentiation or proliferation. The hpg/hpg
mutants cannot produce GnRH, which results in a severe reduction in
pituitary gonadotropins, hypogonadism and low circulating levels of
gonadal steroids. These mice have only slightly reduced lactotrope cell
numbers (26% of the total cells are lactotropes) despite the
established role of estrogen in stimulating lactotrope proliferation
(27). In addition, the lactotropes in hypogonadal mice generated by
transgene ablation of gonadotropes are only reduced to 29% of
wild-type levels (28). The slight reduction in lactotropes in
hpg/hpg and T4-treated
Gsu-/- mutants is probably a secondary
effect due to the lack of normal pubertal maturation during which
estrogen stimulates PRL proliferation (29). These data demonstrate that
an intact pituitary-thyroid axis is essential for the differentiation
or proliferation of PRL-producing cells. Although all of these data
suggest that thyroid hormone is more important for initiating
lactotrope differentiation than gonadal steroids, recent studies
suggest that 2 months of estrogen treatment can stimulate lactotrope
differentiation in thyroid hormone-deficient
Gsu-/- mice (15).
The impaired differentiation of lactotropes in the context of
hypothyroidism is somewhat surprising, given that TRH stimulates PRL
production and lactotrope proliferation (30). The lack of thyroid
hormone might be expected to cause chronic elevation of TRH and
eventually lead to prolactinomas. Instead, the hyt and
Gsu mice both have profound deficiencies in lactotropes.
Thyroid hormone may be required as a priming event to set up the TRH
responsiveness of lactotropes or to regulate expression of a gene that
promotes lactotrope differentiation. Another possibility is that
lineage relationships between the cells exert an influence during
ontogeny that is not evident in mature animals.
Transgene ablation studies have supported the derivation of lactotropes
from somatotropes (26, 31) via an intermediate cell type that produces
both PRL and GH (26). Severe mutations in Pit1 ablate three
cell types, thyrotropes, somatotropes, and lactotropes (32, 33),
supporting the idea that these three cells arise from a common
precursor. Although intermediate cells expressing both GH and TSH have
been observed (34), ablation of thyrotropes by diphtheria toxin had no
impact on somatotrope differentiation, nor does ablation of
somatotropes effect thyrotropes (17, 26, 31). This suggests that
establishment of a normal component of somatotropes and lactotropes
does not require functional thyrotropes (17). On the surface, the
apparent independence of the thyrotrope and somatotrope-lactotrope
lineages in ablation experiments appears in conflict with the
dependence of lactotropes on functional thyrotropes in
Gsu-/- mice. Perhaps, recruitment of
thyrotropes from a common precursor pool depletes the pool available
for generation of lactotropes. As lactotropes are thought to be derived
primarily from somatotropes, it is reasonable to expect that the
consequences for lactotropes would be more severe than those for
somatotropes. Further studies will be required to understand the
mechanism of thyroid hormone stimulation of lactotrope differentiation
and the ability of estrogen replacement to stimulate lactotropes in
thyroid hormone-deficient animals (15).
-Subunit is not required for lactotrope differentiation
-Subunit is expressed early in anterior pituitary development,
several days before the expression of the ß-subunits (35, 36).
-Subunit protein is secreted as a monomer with a different
glycosylation pattern than when it is heterodimerized with ß-subunit
(37). Free -subunit acts as an inducer of lactotrope differentiation
and PRL secretion (6, 25, 38, 39). Consistent with the idea that
-subunit has growth-stimulating properties, the -subunit crystal
structure reveals that it has a cysteine knot tertiary structure
similar to nerve growth factor, transforming growth factor, and
platelet-derived growth factor (40). The deficiency in lactotropes
in Gsu-/- mice initially supported the idea
that -subunit might be critical for lactotrope differentiation. We
have demonstrated that lactotropes can be rescued efficiently by
T4 administration even though the
Gsu-/- mice are hypogonadal and estrogen
deficient. Because the thyroid hormone-treated mice are genetically
unable to produce -subunit, it is clear that free -subunit is not
necessary for the differentiation and proliferation of lactotropes.
Although these experiments demonstrate that -subunit is not
required, we cannot rule out the possibility that -subunit normally
plays a role in stimulating lactotrope differentiation. Many growth
factors and peptides, including nerve growth factor, transforming
growth factor, cytokines, and fragments of POMC, have also been shown
to effect lactotrope differentiation (41, 42, 43, 44, 45, 46). Thus, alteration in the
synthesis or secretion of these or other factors in the mutant mice may
compensate for the lack of -subunit. Direct replacement of
-subunit will be necessary to determine whether it is sufficient for
stimulation of lactotropes in hypothyroid, hypogonadal mice.
Gonadotropes need thyroid hormone for feedback
responsiveness
It has been well established that hyperplasia and hypertrophy of
gonadotropes occur in response to gonadectomy (47, 48). Presumably,
gonadectomy results in the loss of gonadal steroid feedback to the
pituitary, which causes an increase in the synthesis and secretion of
LH and FSH and an expansion in gonadotrope cell area (47). GnRH-treated
hpg mice also exhibit enlarged gonadotrope cells (49). In
contrast, the lack of functional LH and FSH in
Gsu-/- mice did not cause gonadotrope
hypertrophy. However, T4-treated
Gsu-/- mice exhibited remarkable
gonadotrope hypertrophy. This suggests that T4 is required
to establish the sensitivity of gonadotropes to feedback regulation by
gonadal steroids.
The ovaries and uteri of Gsu-/- females
were responsive to gonadotropin and estrogen treatments, respectively
(data not shown). The ovaries of older
Gsu-/- females contained hemorrhagic cysts.
It is well established that an iodine-poor diet induces cystic ovaries;
thus, the cysts in the Gsu mutants probably resulted from
the hypothyroid state (50, 51, 52).
Conclusion
Our studies with the Gsu-/- mice have
demonstrated that -subunit is not required for pituitary lactotrope
differentiation or PRL production. Instead, T4 is critical
for developing a normal component of PRL and GH cells. T4
also appears to be important, directly or indirectly, for priming the
responsiveness of gonadotropes to steroid hormone feedback. The
mechanism for T4 action as a permissive factor for these
three pituitary cell types will be an important area for future
investigation.
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Acknowledgments |
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Gsu-/- mice and helpful discussions. TSH
RIAs were performed in the laboratory of Dr. Douglas Forrest. We are
grateful to those who provided cDNA probes and to the Hormone Pituitary
Program for antibodies and other critical reagents. |
Footnotes |
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2 Current address: Pennsylvania State University College of Medicine,
Mail Code H060, 500 University Drive, P.O. Box 850, Hershey,
Pennsylvania 17033-0850.
3 Current address: Department of Genetics, Case Western Reserve
University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4955.
4 Current address: Department of Biology, 446 Crawford Hall, Eastern
Michigan University, Ypsilanti, Michigan 48197.
5 Current address: National Human Genome Research Institute, 49
Covent Drive, Bethesda, Maryland 20892-4472.
Received June 24, 1998.
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