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-Subunit Promoter/Simian Virus 40 T-Antigen Fusion Gene1
Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Professor Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter (inh-). The mice develop
metastasizing gonadal tumors, of granulosa/theca or Leydig cell origin,
with 100% penetrance by the age of 5–8 months. In the present study,
we examined whether the appearance and growth of the gonadal tumors are
dependent on gonadotropins. Gonadotropin suppression was achieved
either by treatment of 3-month-old mice for 2–3 months with a GnRH
antagonist (Cetrorelix, SB-75), or by cross-breeding the TG mice to the
genetic background of the gonadotropin-deficient hypogonadal mutant
mouse (hpg). Gonadal tumor growth was clearly inhibited
by SB-75 treatment in one of the TG mouse lines (IT6-M), as indicated
by the absence of macroscopically visible tumors and by reduced gonadal
weights. Despite the suppressed gonadotropin secretion and Tag
expression, hyperplasia of testicular Leydig, and ovarian stromal cells
persisted in some of the treated mice. In another TG mouse line
(IT6-F), with more aggressive tumorigenesis, the SB-75 treatment only
partially inhibited gonadal tumor growth. None of the hypogonadotropic
TG mice, homozygous for the hpg mutation, developed
gonadal tumors. Their gonadal histology was indistinguishable from that
of the non-TG hpg mice, suggesting total inhibition of
gonadal tumorigenesis in the absence of gonadotropin stimulation. Tag
expression and Leydig cell hyperplasia were apparent already in the
postnatal TG mice but absent in those TG mice homozygous for the
hpg mutation. In conclusion, the present results
indicate that the gonadal tumorigenesis in our TG mouse model starts in
early age as hyperplasia in specific somatic cells. Both this, and the
subsequent malignant tumor growth, are gonadotropin dependent. A
sufficient level of Tag expression, a prerequisite for gonadal
tumorigenesis, only occurs upon gonadotropin stimulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibin has an established role in the suppression of pituitary FSH
synthesis and secretion, and it has been suggested that it could
function as a physiologic defense mechanism against elevated
gonadotropin levels (10). This is supported by the finding that a
number of ovarian and testicular tumors are associated with elevated
levels of serum immunoreactive inhibin and reduced levels of serum
gonadotropins (11, 12, 13). Importantly, targeted disruption of the mouse
inhibin -subunit gene resulted in formation of aggressive gonadal
tumors, indicating that inhibin is a tumor-suppressor molecule in these
tissues (14, 15).
We have produced a transgenic (TG) mouse model for tumorigenesis of
gonadal somatic cells using a 6-kb fragment of the mouse inhibin
-subunit promoter (inh-) fused with the Simian virus 40 T-antigen
(Tag) coding sequences (16, 17). Gonadal tumors originating from Leydig
or granulosa/theca cells develop in two established TG mouse lines
(IT6-M and IT6-F) with 100% penetrance by the age of 5–8 months.
The aims of the present study were to investigate whether the genesis and growth of the gonadal tumors in the TG mice are regulated by gonadotropins. Two approaches were used to achieve suppression of gonadotropins: 1) long-term (2–3 months) treatment of adult (3-month-old) TG mice with a GnRH antagonist, and 2) cross-breeding of the TG mice into the genetic background of the gonadotropin-deficient hypogonadal mutant mouse (hpg) (18). In addition, the Tag/hpg double mutant mice allowed us to study the gonadotropin dependence of tumorigenesis in the early postnatal period.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Tag transgene, as earlier
described (16). The heterozygous hpg mice for cross-breeding
studies were purchased from the Jackson Laboratory (Bar Harbor, ME).
Genotyping of the mice was performed from tail DNA by PCR as earlier
described (16, 19). The mice were housed 4–6 per cage, after weaning
at the age of 21 days, in a room with controlled light (12-h light,
12-h dark) and temperature (21 ± 1 C). They were fed with mouse
chow SDS RM-3 (Special Diet Service; E, soy-free; Whitham Essex, UK)
and tap water ad libitum. The mice were specific
pathogen-free, and they were routinely screened for common mouse
pathogens. All the procedures using mice were approved by the
University of Turku Ethical Committee on Use and Care of Animals.
GnRH antagonist treatment.
Heterozygous TG mice were used in
the experiments. Five males and 7 females of the IT6-M mouse line, and
5 males and 6 females of the IT6-F line, were injected sc every 84
h with 10 mg/kg BW of the GnRH antagonist Cetrorelix acetate (SB-75;
Asta Medica AG, Frankfurt am Main, Germany) in 5% mannitol. The
control groups receiving similar injections of 5% mannitol consisted
of age-matched TG mice (IT6-M: 5 males, 8 females; IT6-F: 3 males, 5
females). SB-75 treated non-TG mice served as controls for the efficacy
of the treatment. The treatment was started at the age of 3 months. TG
mice of the IT6-M line were treated for 2.5–3 months, whereas those of
the IT6-F line received injections only for 2 months because of the
aggressive gonadal tumor formation in the IT6-F controls. The animals
were killed 3 days after the last injection.
hpg experiment.
In the first mating, heterozygous TG males
of both IT6 lines were crossbred with females heterozygous for the
hpg mutation (HT females). TG males heterozygous for the
hpg mutation (Tag/HT males) derived from the first mating
were further crossbred with HT females to produce the hypogonadal
transgenic (Tag/hpg) mice used in this study (1 IT6-M
Tag/hpg female, and 5 IT6-F Tag/hpg females; 2
IT6-M Tag/hpg males, and 2 IT6-F Tag/hpg males).
Tag/HT litter mates (24 males and females), and hpg mice (9
females, 7 males) served as control animals. All mice were killed at
the age of 6 months.
The mice were anesthetized with Avertin (20) and body weights were recorded. Blood samples were collected in heparinized syringes by cardiac puncture. The blood was allowed to clot overnight at 4 C, and centrifuged (300 x g) at room temperature to separate serum. The sera were stored at -20 C until analyzed. The gonads and adrenal glands were dissected out, weighed, and snap-frozen in liquid nitrogen, or fixed in Bouin’s solution. Seminal vesicles and uteri were weighed. Various other tissues (pituitary, liver, lungs, kidney, uterus, spleen, thymus, heart, brain, and submandibular gland) were taken for RNA or histological analyses.
Experiments with neonatal and developing mice.
Appropriate
breedings and cross-breedings were carried out to obtain wild-type
mice, and those heterozygous for the inh-/Tag transgene, homozygous
for the hpg gene, and those with the two latter genotypes
combined. The mice born from the breedings were killed at the age of 1
or 5 days, and the gonadal tissues were weighed and processed for
histology and RNA preparation as described above. Genotyping of the
animals was carried out by PCR as detailed above, and samples from
animals with the selected genotypes were used for further study.
Morphometric determination of Leydig cell volume density was carried
out as described below. In addition, several Tag expressing mice were
killed at weekly intervals up to 8 weeks of age. Their gonads were
processed for RT-PCR analysis (see below) of Tag messenger RNA (mRNA)
expression.
Hormone measurements
FSH of the pituitary homogenates was measured by a
double-antibody RIA (NIDDK; Bethesda, MD) as described earlier (21).
The hormone preparation was radioiodinated with sodium
[125I]-iodide (IMS 300; Amersham, Buckinghamshire, UK)
using the chloramine-T method (22). LH of the sera and pituitary
homogenates was measured by a supersensitive immunofluorometric assay
(Delfia; Wallac OY, Turku, Finland) developed in our laboratory for rat
LH (23). The protein content of pituitary homogenates was measured by
the Bradford method (24). Progesterone, testosterone, and estradiol
were measured from diethyl ether extracts of the sera by RIAs as
described earlier (25, 26, 27). Inhibin immunoreactivity (also monitoring
the inhibin subunits) in the sera was measured using an RIA kit (from
Dr. G. Bialy, NIH, Bethesda, MD) (28). Two-site ELISAs were used for
specific measurements of the inhibin A or inhibin B dimers (29, 30).
RT-PCR/Southern hybridization
Total RNA from various tissues was isolated by the TRIzol
reagent (Life Technologies, GIBCO-BRL, Glasgow, Scotland) according to
the instructions of the manufacturer. The transgene expression was
studied by RT-PCR. Two micrograms of total RNA were reverse transcribed
by AMV Reverse Transcriptase (F-570L; Finnzymes, Espoo, Finland) and
amplified using Dynazyme thermostable recombinant DNA-polymerase
(Finnzymes) in the same reaction tube (31) in a thermal cycler. In the
first step, cDNA was synthesized in a 10-min incubation at 50 C. Tag
PCR consisted of 40 cycles of the following steps: denaturation for 1
min at 96 C, annealing for 1 min at 56 C, extension for 1.5 min at 72
C. The primer pairs used have been described earlier (16). Forty
percent of the PCR product was resolved on a 1% agarose gel and
transferred onto nylon membrane (Hybond-N, RPN 303N; Amersham). The
specificity of the RT-PCR products was determined by hybridizing the
membranes with a nested oligonucleotide end-labeled with
[32P]
-ATP (Amersham). Hybridization was detected by
autoradiography using Kodak-film (X-Omat AR diagnostic film XAR5,
Eastman-Kodak, Rochester, NY).
Northern hybridization
Total tissue RNA was isolated by the guanidium
isothiocyanate/CsCl method (32). Twenty micrograms of denatured total
RNA were resolved on a 1% denaturing agarose gel and transferred onto
nylon membrane (Hybond-N). The membranes were hybridized with cDNAs for
the rat inhibin -subunit (1.3 kb insert; donated by Dr. H. Meunier,
The Salk Institute, La Jolla, CA) (33) and SV40 Tag (2.7 kb). The
probes were labeled with [32P]-CTP by the random
priming method using Prime-a-Gene kit (Promega, Madison, WI).
Hybridization and washing of the membranes were performed as previously
described (34). Hybridization signals were visualized by
autoradiography using Kodak film.
Histology and immunocytochemistry
Bouin-fixed paraffin sections (5 µm thick) of gonads were
stained with hematoxylin/eosin for histological analysis. Sections of
the same tissues were used for immunocytochemical staining with a
rabbit polyclonal anti-SV40 Tag antibody (1:500 to 1:5000 in
PBS)(kindly donated by Dr. D. Hanahan, University of California, San
Francisco, CA) (35). The antigen-antibody complexes were visualized
with the immunoperoxidase technique (Vectastain Elite ABC kit, Vector,
Burlingame, CA).
Morphometry
Four microscopic fields (40 x magnification) per sample
were selected at random, and counted for the number of Leydig cells.
The total number of cells counted was considered to correlate with the
volume density of Leydig cells in a given sample.
Statistical analysis
The data were analyzed by nonparametric Mann-Whitney Rank-test
using the Macintosh version of the StatView program (Abacus Concepts,
Inc., Berkeley, CA). P values less than 0.05 were regarded as
statistically significant. The numeric data are presented as mean
± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The majority of the control IT6-M TG mice had
developed gonadal tumors (females 5/8; males 5/5), and the gonadal
weights were greater in comparison to the non-TG controls. In the IT6-F
TG mouse line, SB-75 treatment was clearly less effective in inhibiting
tumor growth because 3/6 females and 2/5 males had developed
macroscopically discernible tumors. Every age-matched control mouse of
the IT6-F TG line had developed gonadal tumors (females 5/5; males
3/3). SB-75 treatment did not significantly affect the ovarian weights
of the IT6-F females, whereas the testicular and seminal vesicle
weights of the males of the same line were reduced (Table 1).
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).
As expected, the majority of the Tag/HT mice developed gonadal tumors,
resulting in elevated gonadal weights (Table 1).
Gonadal histology and immunohistochemistry
Histological analysis of testis sections of the SB-75 treated
IT6-M and IT6-F males revealed clear Leydig cell hyperplasia, although
macroscopically discernible tumorigenesis was absent (Fig. 1A). These cells did not show immunostaining for Tag,
whereas a clear positive reaction was observed in TG control testes
(results not shown). Similar Leydig cell hyperplasia was not detected
in SB-75 treated non-TG testes (Fig. 1B) (testis weight: 65.3 mg,
vs. SB-treated TG testis in 1A: 61.4 mg). Despite the SB-75
treatment, full spermatogenesis was observed in some TG and non-TG
testes (results not shown). Ovarian histology of the SB-75 treated
females of both TG lines showed great variation. The majority of the
ovaries were devoid of macroscopically visible tumors, whereas stromal
hyperplasia was detectable in some of them (Fig. 1C). All stages of
folliculogenesis were present (data not shown). Some SB-75 treated
ovaries of the IT6-F line showed staining for anti-Tag in the
granulosa, stromal, and thecal cells (Fig. 1E), whereas in the SB-75
treated IT6-M ovaries, no immunostaining was detected (data not
shown).
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, A and B). No large antral follicles or corpora
lutea were detected. Testicular histology of the Tag/hpg and
hpg males revealed spermatogenic arrest and underdevelopment
of the interstitial compartment (Fig. 2, C and D). Tumorigenesis and
expression of the Tag protein were totally absent in the
Tag/hpg ovaries (results not shown), whereas in the Tag/HT
ovaries, immunostaining for Tag was detected in the tumor tissue, in
perifollicular theca cells and in granulosa cells of large antral
follicles (Fig. 2E). Leydig cell hyperplasia and expression of the Tag
protein were absent in the Tag/hpg testes, whereas in the
Tag/HT testes, Tag immunostaining was detected in the interstitial
tumor tissue (data not shown).
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and 3). The serum FSH levels are not shown because the
concentrations in all groups were low and inconclusive. Pituitary LH
concentrations were significantly reduced in the SB-75 treated IT6-M
males and females (Tables 2 and 3), whereas no differences were found
between SB-75 treated and control mice in the IT6-F line. In contrast,
serum LH concentrations were significantly reduced only in the SB-75
treated IT6-F females (Tables 2 and 3). In the SB-75 treated TG
females, serum progesterone and estradiol concentrations were not
significantly reduced, nor did the treatment alter serum immunoreactive
inhibin levels, as compared with the respective control females (Table 2). In SB-75 treated TG males, serum testosterone concentrations were
significantly reduced, whereas serum progesterone, estradiol (not
shown) and immunoreactive inhibin levels were not altered by the
treatment (Table 3). All control groups of TG mice had elevated serum
levels of immunoreactive inhibin as measured by RIA (Tables 2 and 3).
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and 3). The Tag/HT females
had a high content of pituitary FSH, though with great variation.
Pituitary LH content was reduced in both sexes of the
Tag/hpg and hpg mice (Tables 2 and 3). Serum LH
concentration was significantly reduced in the Tag/hpg
females and in hpg males (Tables 2 and 3). Progesterone
concentration was significantly reduced in the Tag/hpg
females, compared with the Tag/HT females which had elevated levels of
this hormone in comparison to control females (Table 2). Also the
Tag/HT males had elevated serum progesterone as compared with control
males (Table 3). Serum estradiol concentration showed large variation
in the sera of the Tag/hpg females, whereas it was reduced
in the hpg females (Table 2). Testosterone concentration was
significantly reduced in the sera of the Tag/hpg and
hpg males (Table 3). Importantly, the immunoreactivities for
inhibin B or inhibin A could not be detected in the sera of the
Tag/hpg females (only the inhibin B data are shown), nor in
the sera of the hpg mice of either sex, as measured by ELISA
(Tables 2 and 3). The Tag/HT females had a lower concentration of
inhibin B in serum, compared with control females (Table 2).
Expression of SV40 Tag and endogenous inhibin mRNA
The expression levels of SV40 Tag and inhibin mRNAs were
clearly reduced after SB-75 treatment in ovaries of the IT6-M line, and
marginally in the IT6-F line, as analyzed by Northern hybridization
(Fig. 3). Expression of SV40 Tag mRNA was detected in
every tissue of Tag/hpg and Tag/HT mice, except in the
epididymis, as analyzed by RT-PCR/Southern blotting (Fig. 4). All ovarian and testicular tissue samples collected
between day 1 and week 8 post partum displayed clear Tag mRNA
expression by RT-PCR (data not shown).
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. Despite some
missing observations, we can draw clear conclusions on the data. The TG
expression slightly increased testicular weights at both ages but had
no effect on the 5-day ovaries. The hpg mutation alone
reduced on day 5 the testicular weights, but not those of the ovaries.
The Tag/hpg double mutant mice had larger testis weights
than the hpg mice (day 1). The volume density of testicular
Leydig cells was increased by Tag expression but inhibited by the
hpg mutation, which alone had no effect on this
parameter.
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). This
indicates that gonadotropin action, at least in the postnatal testis,
is able to up-regulate Tag expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter. The suppression of gonadotropin
secretion by GnRH antagonist (SB-75) treatment between 3 and 5–6
months of age resulted in reduced growth of gonadal tumors, whereas
gonadal tumorigenesis was totally abolished in the hypogonadotropic TG
mice (Tag/hpg), practically devoid of circulating
gonadotropins from the fetal period onwards. Gonadal tumorigenesis was not totally eliminated by the GnRH antagonist treatment because histology showed interstitial cell hyperplasia in some testes, and stromal hyperplasia in some ovaries of mice of the IT6-M line. Tag protein could not be detected in the hyperplastic cells, suggesting low expression levels of the transgene, and correspondingly, the expression level of Tag mRNA was reduced in the SB-75 treated gonads. Some of the SB-75 treated IT6-F mice developed macroscopical gonadal tumors, which could reflect the previously documented more aggressive, and probably less gonadotropin-dependent, tumorigenesis of this TG mouse line (17).
Besides the reduction of gonadal and accessory sex gland weights,
pituitary LH content of the IT6-M mice, serum LH concentration of IT6-F
females, and serum testosterone concentration of TG males of both lines
were significantly reduced by the SB-75 treatment, indicating the
effectiveness of the GnRH antagonist. In contrast, the pituitary FSH
concentration, as well as those of serum progesterone, estradiol, and
inhibin were unaltered in the SB-75 treated TG mice, indicating only
partial suppression by the treatment. The reason why the pituitary FSH
levels were not suppressed in the tumor mice is apparently due to the
fact that the tumor tissues mainly produce free inhibin -subunits
(36). Other studies with SB-75 have reported more pronounced
suppression of serum LH, FSH, and testosterone levels (37). The
discrepancy can be explained by the infrequent administration of the
antagonist (twice a week) and by the fact that the samples were
collected 3 days after the last injection, allowing time for the
pituitary to recover. Furthermore, SB-75 is more effective in the mouse
when administered by continuous delivery systems, such as microcapsules
(38). Therefore, the present results with the GnRH antagonist show that
partial suppression of gonadotropins suppresses the progression of
gonadal tumors. It remains to be studied which one of the two
gonadotropins is more important for the tumor growth.
As expected, the pituitary contents of FSH and LH showed clear suppression in the Tag/hpg and hpg mice. Serum FSH measurements yielded inconclusive results due to low levels, whereas the serum LH concentrations were significantly reduced in the Tag/hpg females and hpg males. Hpg mice have been reported to have minimal production of testosterone and estradiol, whereas serum progesterone concentration, apparently of adrenal origin, is within the normal range (39, 40, 41). We could detect a significant reduction only in serum testosterone concentration of Tag/hpg and hpg males, in serum progesterone concentration of Tag/hpg females, and in serum estradiol concentration of hpg females. The Tag/HT mice had elevated serum levels of progesterone possibly produced by gonadal tumor cells, as we have shown before (16, 17).
None of the Tag/hpg mice developed gonadal tumors, and
externally, these mice had the hpg phenotype (18). Moreover,
the gonadal weights in both sexes of Tag/hpg mice, and the
uterine weights in females, were drastically reduced. The suppressive
effect of the hpg mutation on gonads was apparent already in
the neonatal period, when the Leydig cell hyperplasia was absent in the
Tag/hpg double mutants. At histological examination, the
Tag/hpg and hpg gonads could not be
distinguished. The Tag protein was not detected by immunohistochemical
staining in the Tag/hpg gonads, whereas it was expressed in
the gonadal tumors of Tag/HT mice, as well as in theca and granulosa
cells of late antral follicles of the Tag/HT females, consistent with
the reported expression pattern of the endogenous inhibin gene in
mouse ovary (42). On this basis, we suggest that the lack of gonadal
tumorigenesis in the Tag/hpg mice, and the suppression of
tumor growth in the SB-75 treated IT6-M mice, are connected to the
dramatically reduced expression of the Tag protein because low
gonadotropin levels have been reported to down-regulate the inhibin
gene expression (43, 44, 45, 46), and thereby also the expression of
inh-/SV40 Tag transgene.
We have previously detected adrenal gland tumorigenesis in the TG mice after prepubertal gonadectomy (36), but not in gonad-intact TG mice, which suggests the presence of gonadal factors inhibiting adrenal tumorigenesis. The suppression of circulating gonadotropin levels in the SB-75 treated TG mice and the Tag/hpg model resulted in a state of functional gonadectomy, and consequently, in deprivation of the putative gonadal factors inhibiting adrenal tumorigenesis. We expected that adrenal tumors would appear in these two models. However, because this was not the case, the adrenal tumorigenesis may be dependent on some other changes (elevated gonadotropins?) related to surgical gonadectomy. The mechanism of the adrenal tumorigenesis will be studied further.
The present TG mouse model is very similar to the inhibin knock out
mouse (14, 47), which also develops gonadal tumors that are prevented
by gonadotropin suppression. Likewise, gonadectomy results in
adrenocortical tumorigenesis in both models (15, 44). In the knock-out
model the gonadotropin effect on tumorigenesis must be independent and
not through stimulation of inhibin- expression. Hence, two
independent factors, absence of inhibin and presence of gonadotropins,
trigger tumorigenesis in this model. In our TG model, the situation is
different because the gonadotropins most likely promote tumorigenesis
by stimulating Tag expression. The fact that testicular weights of the
Tag/hpg mice were higher than those of the hpg
mice neonatally suggests that some Tag effects are possible
without concomitant gonadotropin action. There are also data on gonadal
tumorigenesis in TG mice overexpressing LH (48). Hence, besides
stimulating inhibin- (and Tag) expression, gonadotropins can also
independently promote gonadal tumorigenesis.
The small size of the Tag/hpg gonads did not allow Northern
hybridization analysis for Tag mRNA expression. In the Tag/HT and
Tag/hpg mice, Tag mRNA was found in every tissue analyzed by
RT-PCR/Southern hybridization, indicating that hypogonadotropism does
not eliminate the low basal, nontissue-specific expression of the
transgene. A recent report on the expression level of the inhibin
-subunit mRNA showed no differences between hpg and
control ovaries, indicating constitutive, i.e.
gonadotropin-independent, expression of the inhibin gene in mouse
ovary (49). This suggests that the expression level of endogenous
inhibin and that of Tag mRNAs could remain high in
Tag/hpg ovaries. However, the Tag/hpg females did
not produce SV40 Tag-induced ovarian tumors, nor did they secrete
inhibin. This suggests that, in the present mouse model, the inhibin
-subunit promoter does not display, without gonadotropin
stimulation, sufficient activity for expression of functionally
meaningful amounts of SV40 Tag protein in the Tag/hpg ovary.
The same was apparent from the absence of Tag expression in the
interstitial tissue of the neonatal Tag/hpg mice.
In conclusion, both gonadal tumorigenesis and tumor progression in the
inh-/SV40 Tag TG mice are dependent on circulating gonadotropin
levels. Low gonadotropin levels throughout life in the
Tag/hpg mice totally prevent tumorigenesis, whereas
suppression of gonadotropins by GnRH antagonist treatment suppresses
tumor growth.
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Acknowledgments |
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Footnotes |
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Received February 4, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Inhibin is a tumour-suppressor gene with a gonadal
specificity in mice. Nature 360:313–319[CrossRef][Medline]
-subunit promoter/Simian virus T-antigen
fusion gene: characterization of ovarian tumors and establishment of
gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616–627[Abstract]
-subunit promoter directs SV40 T-antigen to Leydig cells of
transgenic mice. Mol Cell Endocrinol 119:135–146[CrossRef][Medline]
- and ß-subunit
complementary deoxyribonucleic acids and expression in the rat testis
and ovary. Mol Endocrinol 1:561–568[Abstract]
-subunit promoter/Simian
virus 40 T-antigen fusion gene: evidence for negative autoregulation of
the inhibin -subunit gene. Mol Endocrinol 10:1667–1677[Abstract]
-subunit
gene during mouse gametogenesis. Differentiation 44:62–68[CrossRef][Medline]
-subunit gene
in vivo and in vitro. Mol Endocrinol 3:29–35[Abstract]
and ß-B
subunit messenger RNAs in immature and adult animals. Endocrinology 127:1097–1104[Abstract]
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S. B. Rulli, A. Kuorelahti, O. Karaer, L. J. Pelliniemi, M. Poutanen, and I. Huhtaniemi Reproductive Disturbances, Pituitary Lactotrope Adenomas, and Mammary Gland Tumors in Transgenic Female Mice Producing High Levels of Human Chorionic Gonadotropin Endocrinology, October 1, 2002; 143(10): 4084 - 4095. [Abstract] [Full Text] [PDF]
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 M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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G. P. Risbridger, J. F. Schmitt, and D. M. Robertson Activins and Inhibins in Endocrine and Other Tumors Endocr. Rev., December 1, 2001; 22(6): 836 - 858. [Abstract] [Full Text] [PDF]
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I. Huhtaniemi and A. Bartke Perspective: Male Reproduction Endocrinology, June 1, 2001; 142(6): 2178 - 2183. [Full Text] [PDF]
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 N. A. Rahman and I. T. Huhtaniemi Ovarian Tumorigenesis in Mice Transgenic for Murine Inhibin {{alpha}} Subunit Promoter-Driven Simian Virus 40 T-Antigen: Ontogeny, Functional Characteristics, and Endocrine Effects Biol Reprod, April 1, 2001; 64(4): 1122 - 1130. [Abstract] [Full Text]
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A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
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Rilianawati, T. Paukku, J. Kero, F.-P. Zhang, N. Rahman, K. Kananen, and I. Huhtaniemi Direct Luteinizing Hormone Action Triggers Adrenocortical Tumorigenesis in Castrated Mice Transgenic for The Murine Inhibin {alpha}-Subunit Promoter/Simian Virus 40 T-Antigen Fusion Gene Mol. Endocrinol., June 1, 1998; 12(6): 801 - 809. [Abstract] [Full Text]
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