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Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622
Address all correspondence and requests for reprints to: Dr. William L. Miller, North Carolina State University, Department of Biochemistry, Box 7622, Raleigh, North Carolina 27695-7622. E-mail: wlmiller{at}bchserver.bch.ncsu.edu
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Introduction |
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-subunit combined with a unique
ß-subunit. Because synthesis of the ß-subunit controls overall FSH
production, knowledge of how FSHß is regulated is vital to
understanding the female reproductive cycle. FSHß is produced in gonadotrope cells of the anterior pituitary, and its synthesis is regulated by hypothalamic GnRH, gonadal peptides (activin, inhibin, and follistatin), and gonadal steroids (progesterone, estradiol, and testosterone). Pulsatile GnRH is required to stimulate FSHß gene expression (2), whereas continuous treatment with GnRH down-regulates FSHß messenger RNA (mRNA) levels (3). Activin increases, whereas inhibin and follistatin decrease FSHß mRNA levels (4). Progesterone exerts a direct negative effect on FSHß gene transcription and FSH secretion in the sheep pituitary (5), but positively regulates FSH secretion in rat pituitary cultures (6). Estradiol represses FSHß gene expression directly at the pituitary level in sheep (5, 7), but has a minimal direct pituitary effect in the rat (8, 9). Testosterone has been shown to decrease the postcastration rise of FSHß mRNA (10), although the suppressive effect of testosterone is indirectly at the hypothalamic level (9).
Due to the lack of an appropriate cell line, it has been difficult to analyze the regulatory elements that confer cell-specific expression and hormonal responsiveness on the FSHß gene. Transient transfection of heterologous cell lines with constructs that contain the ovine FSHß promoter linked to a luciferase reporter gene has been useful in localizing sequences that show responsiveness to progesterone (11), estradiol (12), and GnRH (13), but it is unclear how these sequences help regulate FSHß transcription in gonadotropes in vivo. Furthermore, it has been impossible to identify those sequences necessary for cell-specific expression, because the FSHßLuc constructs are equally active in many heterologous cell lines. Recently, it was discovered that a gonadotrope cell line, LßT2 cells, can express FSHß when treated with activin (14), and this opens the possibility of dissecting FSHß regulation in a fully functional gonadotrope cell line using transfection methods. However, regardless of the in vitro methods used to analyze FSHß regulation, it will ultimately be necessary to verify the results in vivo.
The transgenic mouse model offers a powerful tool to test the physiological importance of promoter elements defined using in vitro transfection studies. Transgenic mice carrying the 10-kb human FSHß gene that contains 4.3 kb of the 5'-promoter region, the entire coding sequence, and 2 kb of 3'-flanking sequence were generated, and the transgene was expressed exclusively in pituitary gonadotropes and regulated similarly to the endogenous mouse FSHß gene (15). However, attempts to use the human FSHß promoter to express a reporter gene specifically in pituitary gonadotropes in transgenic mice have not previously been successful.
Reported herein are data that establish a transgenic mouse model for studying tissue-specific expression of the FSHß gene in vivo and the physiological relevance of FSHß promoter sequences that are defined by in vitro transcriptional regulation studies. Briefly, the data indicate that pituitary-specific expression and all of the physiologically relevant regulatory mechanisms are encoded by sequences in the ovine FSHß (oFSHß) gene between -4741 and +759 bp. In addition, pituitary cultures derived from these transgenic mice provide a convenient in vitro cell system to dissect transcriptional regulation of the oFSHß gene in gonadotropes.
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Materials and Methods |
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In vivo experiments
Transgenic male and female mice 7 weeks old or older were
divided into the following groups (n = 3–6): intact, castrated,
or Lupron treated. Lupron depot (100 µg), a long-acting GnRH agonist
from Abbott Laboratories (Chicago, IL), was given by a
single injection ip. Animals were killed 14 days after gonadectomy or
Lupron injection. Individual pituitaries were collected and assayed for
luciferase activity.
For estrous cycle studies, vaginal smears were taken to determine the stage of the estrous cycle (16). Mice at diestrus were paired with littermates at estrus and killed at 0900 h, and pituitaries were assayed for luciferase activity.
Cell cultures from transgenic mouse pituitaries
Transgenic mice, 7 weeks old or older, were killed, and
pituitaries were dissected. After rinsing in magnesium-free, low
calcium (0.15 mM) HBSS (Life Technologies, Inc., Grand Island, NY) containing 25 mM HEPES
(Sigma, St. Louis, MO), pituitaries were cross-sliced with
two scalpel blades and digested with collagenase type I
(Sigma; 1.5 mg collagenase/ml buffer were used for 25
mouse pituitaries) to yield a dispersed cell population. The
collagenase buffer contained 3% BSA in magnesium-free, low calcium
HBSS with HEPES, and the pituitary slices were incubated with gentle
shaking in collagenase for 1.5 h at 36 C and then treated with
Pancreatin 4x USP (Life Technologies, Inc.), diluted 1:2
in calcium- and magnesium-free HBSS containing 25 mM HEPES,
for 15 min at 36 C. The dispersed cells were washed three times in
medium 199 (Life Technologies, Inc.), which contained 25
mM HEPES and 10% charcoal-treated sheep serum. After the
final wash, cells were resuspended in medium 199 with 25 mM
HEPES, 10% charcoal-treated sheep serum, 60 µg/ml gentamicin
(Sigma), 10 µg/ml insulin (Sigma), 100 U/ml
penicillin (Sigma), and 0.5 µg/ml fungizone (Life Technologies, Inc.). Dispersed cells were plated into 96-well
tissue culture plates (Primaria, Becton Dickinson and Co.,
Lincoln Park, NJ) at a cell density of 30,000–60,000 cells/200
µl·well and allowed to attach for 1–2 days at 37 C under 5%
CO2/95% air before treatment.
Luciferase assays
For in vivo experiments, transgenic mice were killed
using CO2, and whole pituitaries or other tissues
(0.3–1 mg) were snap-frozen in liquid nitrogen before homogenization
in 200 µl cell culture lysis reagent (Promega Corp.).
Cell debris was pelleted by microcentrifugation at 10,000 x
g for 15 sec, and 20 µl cellular lysate were immediately
assayed for luciferase activity using the luciferase assay system
(Promega Corp.). Luminescence was measured as relative
light units (RLU) for 20 sec using a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, San Diego, CA).
Protein concentrations were determined using bicinchoninic acid
(Pierce Chemical Co., Rockford, IL). Luciferase activity
is reported as RLU per mg protein.
For cell culture experiments, 24 h after addition of hormone, media were removed, and 50 µl Passive Lysis Buffer (Promega Corp.) were added and incubated with cells for 15 min. Thirty-five microliters of the lysate were assayed for luciferase activity.
Hormones
Recombinant human follistatin was provided by the National
Hormone and Pituitary Program of the NIDDK. Progesterone and
17ß-estradiol were obtained from Sigma. Recombinant
human inhibin A was a gift from Prince Henry’s Institute of Medical
Research (Clayton, Victoria, Australia) by Dr. A. J.
Mason.
RIA
The FSH levels were measured with reagents provided by the
National Pituitary and Hormone Program of the NIDDK, using a double
antibody method as previously described (17). FSH was
determined in triplicate with mFSH reference preparation (AFP-5308D) as
standard, anti-mFSH antiserum (AFP-1760191) as primary antibody, and
rFSH-I-8 (AFP-11454B) as trace. Intraassay variation was less than
10%, and all samples from one experiment were assayed together in one
assay.
Statistical analysis
Statistical calculations were performed using Prism
(GraphPad Software, Inc., San Diego, CA). For Tables 2
, 3, and 4, a t test was used to determine whether differences
between control and treatment groups were significant. For Table 6,
one-way ANOVA was used to determine whether differences between
hormone-treated and untreated control groups were significant. If
differences were significant (P < 0.05), Dunnett’s
multiple comparison test was then used for post-hoc
evaluation of differences between different hormone-treated groups and
control groups. For Table 2 and Fig. 2, differences among treatments
and founder lines were also analyzed using ANOVA procedures for a
factorial arrangement of treatments using the "proc mixed"
procedure of SAS Institute, Inc. (Cary, NC). Treatment,
founder line, and interaction were considered to be fixed effects, and
litter and culture were considered to be random effects. When
significant differences among treatments or founder lines were
observed, Tukey’s multiple comparison test was used to determine
differences among treatments.
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)
to 66,000,000 RLU/mg protein (line 4088; see Table 1). One line (4089)
had intact promoter sequences, but expression of luciferase in the
pituitary was highly variable, ranging from 3,000–60,000,000 RLU/mg
protein. One other line (8015) did not express luciferase and had a
promoter deletion between -2755 and -3275 bp, which may be critical
for expression or the transgene may have been integrated into a silent
site within the chromatin.
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Tissue specificity of transgene expression
To investigate tissue expression of the transgene, male and female
mice from the six lines that expressed luciferase in the pituitary were
also analyzed for luciferase expression in seven other tissues: brain,
heart, lung, liver, spleen, and gonads. Expression of oFSHßLuc was
confined almost exclusively to the pituitary in five of the six founder
lines (Table 1
). In the sixth line (4090), expression was quite high in
the pituitary, as it was in all other tissues tested (Table 1),
suggesting that the transgene was integrated into a position within the
chromatin that allowed the gene to be widely expressed. Five lines
(lines 7152, 3861, 3854, 3855, and 4088) were established for further
experimentation.
Effects of gonadectomy and chronic GnRH treatment on transgene
expression
To assess whether transgene expression was affected by gonadal
feedback regulation, male and female mice from five founder lines were
castrated, and pituitary oFSHßLuc activity was measured after 2
weeks. Castration of males resulted in 2- to 8-fold increase in
luciferase activity compared with that observed in intact controls
(Table 2). Castration of females caused a
2- to 27-fold increase in luciferase activity compared with that in
intact randomly cycling controls (Table 2). Due to the lack of a
sufficient number of male offspring from line 4088, castration was not
performed with this line. The increase in pituitary luciferase activity
by castration of female mice from line 3861 and male mice from line
3855 did not reach statistical significance. However, given the small
sample sizes for individual founders, the data from all founders were
combined and subjected to further statistical analysis as described in
Materials and Methods. Treatment effects of castration were
significant in both males and females (P < 0.05).
Transgenic mice also received a single injection of Lupron depot (a
long-acting GnRH agonist) to determine whether long-term GnRH treatment
could down-regulate expression of the transgene. Table 3 shows the results of Lupron treatment
on mice from five founder lines. After a 2-week treatment with GnRH,
pituitary luciferase activity was decreased by 51–99% among five
founder lines.
Levels of oFSHßLuc expression during the estrous cycle
It has been shown that the mRNA level and the basal transcription
rate of the FSHß gene vary during the rat estrous cycle, with lowest
levels on diestrus and highest on the morning of estrus (18, 19). In the mouse, serum FSH values are lowest on diestrus and
highest on the morning of estrus (20, 21). To investigate
whether oFSHßLuc expression changes during the mouse estrous cycle,
transgenic mice at diestrus or estrus were killed in the morning (0900
h), and pituitary luciferase activities were compared. Using all five
founder lines, it was found that luciferase activity was increased 3-
to 20-fold on estrus compared with the activity on diestrus (Table 4). It should be noted that the cycle
stages of the female mice used in Tables 1–3 were not identified, and
thus the data represented an average of expression obtained at all
cycle stages.
Expression of oFSHßLuc in transgenic mouse pituitary
cultures
To explore the direct effects of gonadal hormones on oFSHßLuc
expression in the absence of GnRH, primary pituitary cell cultures were
prepared from the transgenic mice. After being cultured in
vitro, transgenic mouse pituitary cells still expressed relatively
high levels of luciferase activity (Table 5). In all five founder lines tested,
luciferase expression in cultures was relatively low the first day
after dispersion and reached its highest levels 3 days after dispersion
[3,000 (line 3861) to 470,000 (line 4088) RLU/50,000 cells·well;
Table 5]. Expression started to decline 4 days after dispersion and
reached a steady state 6 days after dispersion. To compare the
expression of the transgene with that of endogenous mouse FSH, culture
media were collected every 2 days and assayed for FSH using RIA. The
data in Fig. 1 show the similarity of
changes in oFSHßLuc expression and FSH secretion from mouse pituitary
cultures of the founder line 7152, which was representative of all five
founder lines tested. Note that the value for FSH secretion represents
secretion over the 2 preceding days, whereas luciferase activity
represents luciferase expression at the exact time of cell harvest. It
should be noted that the cultures continue to secrete significant
amounts of FSH and express luciferase after day 4 in culture,
indicating that the cultures were still healthy.
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). The
data from all founders were also combined and subjected to further
statistical analysis as described in Materials and Methods.
Results from all founder lines were presented in Fig. 2A, and treatment effects of follistatin
and inhibin were significant. To show that endogenous mouse FSH was
also regulated by follistatin and inhibin, media were collected from
cultured mouse pituitary cells at the time of harvest and assayed for
FSH. Figure 2B shows that within 24 h, follistatin and inhibin
significantly decreased FSH secretion by 71% and 65%,
respectively.
To determine whether progesterone can repress oFSHßLuc transgene
activity or endogenous production of mouse FSH, pituitary cells
cultured from transgenic mice were treated with 100 nM
progesterone for 24 h. Culture media were collected and assayed
for FSH, and cells were harvested for luciferase activity. Progesterone
significantly decreased luciferase expression by 40–51% in lines
3861, 3854, and 3855 (Table 6), but did not significantly decrease FSH
secretion from the mouse pituitary cultures (Fig. 2B). The luciferase
data from all founders were also combined and subjected to further
statistical analysis as described in Materials and Methods.
Results from all founder lines are presented in Fig. 2A, and treatment
effects of progesterone were significant (P <
0.05).
To determine whether estradiol can regulate oFSHßLuc transgene
activity or mouse FSH production independently of GnRH, transgenic
mouse pituitary cultures from five founder lines were treated with 10
nM estradiol for 24 h. Culture media were then
collected, and cells were harvested for luciferase activity. Secretion
of mouse FSH from the transgenic mouse pituitary cultures was
significantly decreased 21% by estradiol treatment (Fig. 2B). However,
expression of oFSHß was not significantly decreased by estradiol,
except in line 3855 (Table 6). The luciferase data from all founders
(as shown in Fig. 2A) were also combined and subjected to further
statistical analysis as described in Materials and Methods.
Treatment effects of estradiol were not significant (P
< 0.05).
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Five founder lines of transgenic mice (7152, 3861, 3854, 3855, and 4088) were established. Levels of pituitary oFSHßLuc expression varied up to 100-fold among these lines (the 4088 line was highest and 7152 was lowest), which could be due to the different vector backbones used (7152 used the pXP2 vector, whereas all others used the pGL3 vector), copy number, or site of transgene insertion in the mouse genome. Although luciferase expression varied considerably, it was almost exclusively in the pituitary in all transgenic lines, with occasional low expression in the brain (0–5%) and gonads (0–3%). As it has been reported that FSHß mRNA is expressed in the gonads at a much lower level than in the pituitary (23, 24), it was not surprising to find trace levels of oFSHßLuc expression in the gonads and other tissues due to the greater sensitivity of the luciferase assay. All lines tested, regardless of vector backbone or site of genomic integration, responded to physiological and hormonal manipulations with similar changes in pituitary luciferase expression, indicating that the regulation observed with the transgene reflected true regulation of the oFSHß gene.
To further localize the sequences necessary for cell-specific expression, we also generated transgenic mice with truncated oFSHß promoters (-750 to +759 bp and -215 to + 759 bp), but none of them showed luciferase activity in the pituitary or any other tissue. As three and seven lines were generated, respectively, it is unlikely that the lack of expression for these transgenes was merely caused by insertion into silent sites in the chromatin. These results suggested that some sequences required for FSHß expression reside in the distal promoter region between -4741 and -750 bp. It is interesting to note that transgenic mice made with 2.3 kb of 5'-flanking region and 23 nucleotides (nt) of the first exon of the bovine FSHß gene fused to the thymidine kinase reporter gene had equally high expression in pituitary and gonads (25). Apparently this transgene lacked elements that might repress expression in the gonads, indicating that gonadotrope-specific FSHß gene expression requires regulatory elements that enhance expression as well as those that restrict expression to gonadotropes.
Pituitary cell cultures have been used extensively to study hormonal regulation of the FSHß gene in the absence of hypothalamic or gonadal influences. By measuring luciferase activity of pituitary cultures derived from the transgenic mice, it was found that the oFSHßLuc transgene still expressed at high levels in cultures, which allowed us to study oFSHß gene regulation in gonadotropes. Levels of transgene expression were highest on days 2 and 3 of culture, declined on day 4, and reached a lower steady state by day 6. The reason for the decline in luciferase activity could be due to the lack of endogenous FSH-stimulating factors, such as activin or GnRH, normally present in vivo. Whatever the mechanism, it is noteworthy that endogenous mouse FSH secretion followed the same pattern. As secretion of FSH usually reflects expression of the FSHß gene, these results indicated that expression of the transgene mimics that of the endogenous mouse FSHß gene in pituitary cultures. Moreover, follistatin and inhibin both altered transgene activity and endogenous mouse FSH in the same way in culture. It should be noted here that activin also increases oFSHßLuc gene expression dramatically using follistatin- pretreated transgenic mouse pituitary cultures, as shown in the companion paper (26), further demonstrating that the pituitary cultures derived from these transgenic mice can be useful for studying oFSHß gene regulation in gonadotropes by known or novel FSH modulators (27).
Follistatin and inhibin have been reported to decrease steady state levels of FSHß mRNA (4), but it is controversial whether transcription, mRNA stability, or both are affected. Using the transgenic mouse pituitary cultures, we have shown that follistatin and inhibin decreased basal luciferase expression and endogenous mouse FSH secretion to a similar extent. As luciferase expression should reflect transcriptional activity, the data presented here suggest that follistatin and inhibin decrease FSHß mRNA levels at the transcriptional level.
Although the effects of GnRH, follistatin, and inhibin on FSHß gene expression appear to be uniform across species, it has been reported that the effects of progesterone and estradiol are species specific at the pituitary level. In rat pituitary cultures, for example, neither progesterone nor estradiol inhibits FSHß expression, and progesterone can actually increase FSH secretion (6). By contrast, both progesterone and estradiol dramatically suppress FSHß gene expression and secretion in sheep pituitary cultures (5). Pituitary cultures derived from oFSHßLuc transgenic mice provide a unique model for studying the origin of species’ differences in steroid regulation, because the ovine FSHß promoter is being expressed in a mouse gonadotrope. The data reported here show that progesterone decreases oFSHßLuc expression by 40–61%, but did not affect endogenous mouse FSH secretion from transgenic cultures. As in sheep pituitary cultures, both FSH secretion and FSHß gene expression are normally decreased 50–90% by progesterone (5), the results with transgenic cultures suggest that the different progesterone responses seen between mice and sheep may be due to differences at the promoter level.
By contrast, estradiol decreased basal mouse FSH secretion by 21%, but did not significantly change oFSHßLuc expression in pituitary cultures. As estradiol decreases FSH secretion and FSHß gene expression by 60–90% in sheep pituitary cultures (5), the lack of repression of oFSHßLuc expression and the minor decrease observed in mouse FSH secretion suggest that factors made in mouse gonadotropes are different from those made in sheep gonadotropes, rendering the mouse cultures less responsive to negative feedback action by estradiol. This could be due to the nature of the estrogen receptor, coactivator/repressor, or other unknown factors.
By monitoring luciferase expression from pituitaries or pituitary cultures of the oFSHßLuc transgenic mice, transcription of the oFSHß gene can be rapidly measured in vivo or in vitro. To increase the expression of the oFSHßLuc transgene, the first intron of the oFSHß gene was included along with the FSHß promoter [which also includes exon 1 (63 nt) and 62 nt of exon 2). Although it is possible that some regulation is mediated by these exon/intron sequences, in vitro studies have shown that these elements are not involved in regulation by progesterone, estradiol, or GnRH (11, 12, 13).
In summary, we have produced transgenic mice harboring the oFSHßLuc transgene and showed that the oFSHß promoter (-4741 to +759 bp) contains elements sufficient to confer pituitary-specific expression and normal hormonal regulation to this gene. High levels of luciferase expression made it easy to monitor hormonal regulation of the FSHßLuc transgene either in vivo or in pituitary cell cultures. Therefore, these mice provide an in vivo model and a convenient in vitro cell culture system for testing the transcriptional effects of known and/or novel FSHß regulators.
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Acknowledgments |
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Footnotes |
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2 Present address: LabCorp, Research Triangle Park, North Carolina
27709.
3 Present address: Department of Biochemistry and Molecular
Genetics, University of Virginia Health Sciences Center,
Charlottesville, Virginia 22908.
Received September 11, 2000.
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-subunit
messenger ribonucleic acid by gonadotropin-releasing hormone and
estrogen in the sheep pituitary. Neuroendocrinology 50:321–326[CrossRef][Medline]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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H.-J. Huang, J. Sebastian, B. D. Strahl, J. C. Wu, and W. L. Miller Transcriptional Regulation of the Ovine Follicle-Stimulating Hormone-{beta} Gene by Activin and Gonadotropin-Releasing Hormone (GnRH): Involvement of Two Proximal Activator Protein-1 Sites for GnRH Stimulation Endocrinology, June 1, 2001; 142(6): 2267 - 2274. [Abstract] [Full Text] [PDF]
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H.-J. Huang, J. C. Wu, P. Su, O. Zhirnov, and W. L. Miller A Novel Role for Bone Morphogenetic Proteins in the Synthesis of Follicle-Stimulating Hormone Endocrinology, June 1, 2001; 142(6): 2275 - 2283. [Abstract] [Full Text] [PDF]
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