Overexpression of Mouse Follistatin Causes Reproductive Defects in Transgenic Mice
Qiuxia Guo,
T. Rajendra Kumar,
Teresa Woodruff,
Louise A. Hadsell,
Francesco J. DeMayo and
Martin M. Matzuk
Departments of Pathology (Q.G., T.R.K., M.M.M.), Cell Biology
(L.A.H., F.J.D., M.M.M.), and Molecular and Human Genetics (M.M.M.)
Baylor College of Medicine Houston, Texas 77030
Departments of Medicine (T.W.) and Neurobiology and Physiology
and The Center for Reproductive Sciences Northwestern
University Chicago, Illinois 60611
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ABSTRACT
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Follistatin is an activin-binding protein that can
act as an activin antagonist in vitro. Follistatin also
binds heparin sulfate proteoglycans and may function as a reservoir for
activins in vivo. In the mouse, follistatin mRNA is first
detected in the deciduum on embryonic day 5.5 and later in the
developing hindbrain, somites, vibrissae, teeth, epidermis, and muscle.
We have previously shown that follistatin-deficient mice have numerous
embryonic defects including shiny, taut skin, growth retardation, and
cleft palate leading to death within hours of birth. To further define
the roles of follistatin during mammalian reproduction and development,
we created gain-of-function mutant mice in which mouse follistatin is
overexpressed. The mouse metallothionein (MT)-I promoter was
placed upstream of the six-exon mouse follistatin (FS) gene. To
distinguish wild-type and transgenic follistatin mRNA, the
3'-untranslated region of the mouse follistatin gene was replaced with
the SV40 untranslated and polyA sequences. Three male and two female
founder transgenic mice were produced, were fertile, and transmitted
the transgene to offspring. Northern blot analysis demonstrated that
the transgene mRNA was expressed at varying levels in the livers of
offspring from four of five of the transgenic lines and was expressed
in the testes in all five lines. In MT-FS line 4, which had the highest
expression of the transgene mRNA in the liver, the transgene
transcripts were also present in multiple other tissues.
Phenotypically, the MT-FS transgenic lines had defects in the testis,
ovary, and hair. Mice from MT-FS lines 7 and 10 had slightly decreased
testis size, whereas mice from lines 4, 5, and 9 had much smaller
testes and shiny, somewhat irregular, fur. Histological analysis of the
adult testes from line 5 and 9 males showed variable degrees of Leydig
cell hyperplasia, an arrest of spermatogenesis, and seminiferous
tubular degeneration leading to infertility. Female transgenic mice
from lines 4 and 9 had thin uteri and small ovaries due to a block in
folliculogenesis at various stages. Many of the line 9 female mice
eventually became infertile, and all of the line 4 female mice were
infertile. Suppressed serum FSH levels were seen in only the line 4
transgenic male and female mice, the line with widespread expression of
the transgene. Serum FSH levels were not significantly different in
gonadectomized wild-type and line 5 transgenic male mice despite high
levels of the follistatin transgene mRNA in the liver of these
transgenic mice. These results suggest that follistatin exerts its
effects at the levels of the gonads and pituitary as a local
regulator of activin and possibly other transforming growth factor-ß
family members.
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INTRODUCTION
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Activins and inhibins are structurally related proteins
belonging to the transforming growth factor-ß (TGF-ß) superfamily.
The activins are ß:ß dimers (ßA:ßA, ßB:ßB, ßA:ßB) that
share common ß-subunits with the heterodimeric
:ß inhibins.
Activins and inhibins were initially recognized as gonadal peptides
that stimulate or inhibit FSH production by the pituitary in adult
mammals (1, 2, 3). More recently, two related mammalian activin subunit
genes have been cloned: activin ßC and ßE (4, 5, 6). It is unclear
whether dimers of activin ßC or ßE can regulate FSH synthesis or
whether these subunits can dimerize with an inhibin
subunit.
Similar to inhibin, follistatin, a glycosylated monomeric protein,
suppresses pituitary FSH synthesis and secretion (7, 8, 9, 10). In 1990,
Sugino and colleagues (7) demonstrated that follistatin binds to the
ß-subunits of activin in a 1:1 molar ratio to form an inactive
complex (8). In humans, rats, and pigs, the mature follistatin protein
has two forms, follistatin-288 and follistatin-315, which are generated
by alternative splicing of the follistatin mRNA (9).
Activins and follistatin are expressed in multiple tissues during
mammalian development and are involved in diverse physiological and
developmental processes (1, 2, 9, 11, 12, 13, 14). During rat development,
activins can stimulate proliferation of Sertoli cells in the testis and
regulate folliculogenesis in the ovary (3, 15, 16, 17). Studies in
Xenopus laevis and Oryzias latipes have shown
that activins are potent mesoderm-inducing factors (18, 19).
Overexpression of follistatin in Xenopus laevis blocked
activins effects on mesoderm formation and induced neural tissue
directly (20). However, studies using knockout mice in our laboratory
failed to demonstrate a role of activins, follistatin, or activin type
II receptor in mesoderm formation or neural development. Mice deficient
in activins can survive to birth but demonstrate craniofacial defects
(21). Mice lacking follistatin have multiple defects, including
musculoskeletal and dermatopathological abnormalities, and die within
hours of birth (22). These defects are more widespread than those seen
in activin- or activin receptor type II-deficient mice (21, 23).
In the liver, activin A has been reported to inhibit the initiation of
DNA synthesis in rat hepatocytes in vitro and in
vivo (24). Use of recombinant activin A in vitro or
in vivo induces cell death via apoptosis in rat and murine
hepatocytes (25, 26). Studies in our laboratory using mice deficient in
both inhibin and activin receptor II (ActRII) have demonstrated that
activins, secreted from gonadal tumors, signal through ActRII in
hepatocytes to directly cause the hepatocellular necrosis seen in
-inhibin-deficient mice (27, 28). Intraportal or intravenous
administration of follistatin can block the inhibitory effects of
activins on hepatocytes, thereby accelerating liver regeneration in
partially hepatectomized rats (29, 30).
The generation of mice lacking either inhibin or activin receptor
type II has also confirmed that inhibins and activins are important
regulators of pituitary FSH levels in vivo (23, 31). Mice
lacking inhibin have elevated FSH levels (31) whereas mice lacking
activin receptor type II (23) have suppressed FSH levels. Females
lacking activin receptor type II are infertile due to a block at the
antral follicle stage, whereas males lacking this activin receptor have
small gonads and delayed fertility (23). However, since
follistatin-deficient mice die at birth, similar analyses of the
essential roles of follistatin in the regulation of FSH synthesis and
secretion and its roles in adult reproductive physiology are
impossible.
To further understand the function of follistatin in mammalian
development and reproduction, we created transgenic mice overexpressing
the wild-type mouse follistatin gene product using the mouse
metallothionein (MT)-I promoter. In this report, we show that the
follistatin transgene was expressed in multiple tissues leading to
major defects in the reproductive tract and minor defects in hair
formation. These studies demonstrate that follistatin plays an
important role in reproductive physiology in vivo.
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RESULTS
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Generation of Mice Overexpressing Follistatin and
Expression Analysis of the MT-FS Transgene mRNA
Transgenic mice overexpressing the wild-type mouse
follistatin gene product using the mouse metallothionein-I promoter
(32, 33) (Fig. 1
) were created in this
study. Five founder transgenic mice (three male and two female) were
produced and were fertile. Southern blot analysis of genomic DNA from
offspring from these founder mice demonstrated that all five lines
transmitted the transgene. However, breeding of the line 4 founder male
with wild-type mice demonstrated that the ratio between transgenic and
wild-type mice was much lower than 50% (10 transgene positive
offspring of 80 total). This suggested that the testes (i.e.
germ cells) of this line 4 founder mouse were likely mosaic. As
expected, the integrated transgene copy number was different between
the various lines: line 9 had the highest copy number and line 4 had
the lowest (Fig. 2
and data not
shown).

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Figure 1. The MT-FS Transgene Construct
A 1.8-kb mouse metallothionein-I (mMT-I) promoter fragment (32, 33) was
fused to a 5.1-kb genomic fragment (2, 22) containing the six coding
exons of the mouse follistatin gene. A 973-bp fragment containing the
simian virus 40 (SV40) 3'-untranslated region and polyadenylation
signal were placed downstream of the six coding exons in lieu of the
3'-UTR of the endogenous follistatin gene.
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Figure 2. Southern Blot Analysis of Transgenic Genomic DNA
Transgenic mice were identified by Southern blot analysis using the
973-bp SV40 3'-UTR/poly A fragment as a probe. Offspring from three of
the five transgenic lines are shown from the same hybridization
reaction. The order of lines with respect to number of transgene copies
are approximated as follows: line 9 line 5 > line 10
> line 7 line 4.
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Northern blot analysis was performed to analyze the expression of the
MT-FS transgene in both reproductive and nonreproductive tissues. As
shown in Fig. 3A
, the transgene mRNA was
expressed at varying levels in the livers of offspring from four of
five of the transgenic lines and was expressed in the testes of all
five of the lines. Since liver expression of the MT-FS transgene was
highest in lines 4 and 5, multitissue Northern blot analysis was also
performed on these two lines to determine whether the transgene was
constitutively expressed in any tissues other than the liver and
testes. The MT-FS transgene was also expressed in the skin and brain in
both lines (Fig. 3B
and data not shown). In line 4, there was
additional expression in the heart, lung, spleen, stomach, kidney, and
small intestine. Since overexpression of follistatin in Xenopus
laevis causes defects in mesoderm formation and neurulation, we
also checked whether the follistatin transgene was expressed during
embryogenesis. By Northern blot analysis of total RNA, the MT-FS
transgene was expressed in the livers of all line 5 mice examined at
embryonic day 18.5 (E18.5) (data not shown). RT-PCR analysis of total
RNA from E6.5 and E7.5 line 5 embryos also demonstrated the presence of
the MT-FS transgene transcript (data not shown).

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Figure 3. Follistatin Transgene Expression in Adult Tissues
Total RNA (15 µg) extracted from various tissues of adult transgenic
mice was subjected to Northern blot hybridization using the 973-bp SV40
3'- UTR/poly A fragment as a probe. Total RNAs from a wild-type mouse
were used as negative controls. Equivalent loading of RNA was confirmed
by hybridization with a ribosomal 18S RNA cDNA probe. A, Follistatin
transgene expression in livers and testes from various transgenic
lines. B, Follistatin transgene expression in multiple tissues from a
line 4 transgenic mouse.
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In other mammalian species, endogenous follistatin mRNA is
alternatively spliced at the 3'-end, giving rise to two distinct
protein forms (9, 13). To determine which mRNA and protein forms were
expressed in wild-type and MT-FS transgenic mice, we performed PCR
analysis of RNA derived from either wild-type ovary (the most abundant
source of follistatin) or transgenic testis (line 7). Electrophoretic
and Southern blot analysis of these PCR products demonstrated that both
wild-type and transgene follistatin mRNAs exist as only one species,
encoding the long 343-amino acid precursor form of the mouse protein
(11) (data not shown).
Morphological and Histological Analyses
All of the MT-FS transgenic offspring from the five independent
lines were viable and developed to adults. In contrast to
overexpression of follistatin in Xenopus laevis, in which
mesoderm and neurulation defects are observed, overexpression of
follistatin during early mouse development did not result in any
deleterious effects. Activins have been shown to have an inhibitory
effect on GH production in vitro (1, 34), and therefore, the
MT-FS transgenic mice and littermates were weighed weekly. Similar to
activin ßB (35) or activin receptor type II knockout mice (23), adult
female and male MT-FS transgenic mice did not demonstrate any
statistically significant changes in body weight compared with controls
(data not shown). Since the MT-FS transgene is highly expressed in
liver, and since follistatin and activins are known to effect liver
apoptosis and regeneration (24, 25, 26, 27, 28, 29, 30), we examined the transgenic livers
morphologically and histologically. The liver weights of these
transgenic male and female mice are not statistically different
compared with the liver weights of wild-type littermate controls (data
not shown). Histological analysis of livers from these transgenic lines
also did not show any abnormalities (data not shown).
Newborn mice lacking follistatin (22) or activin ßA (23) have whisker
defects. Prepubertal and adult MT-FS transgenic mice also had gross
abnormalities in the hair. Mice from lines 4, 5, and 9 demonstrate
shiny, somewhat irregular, fur (Fig. 4A
)
and could be distinguished grossly from their littermates as early as 3
weeks of age. Histological analysis of the skin, however, revealed no
obvious cause for this phenotype (data not
shown).

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Figure 4. Morphological, Histological, and Biochemical
Analysis of Wild-Type and MT-FS Transgenic Male Mice
A, Gross analysis of the fur of a 4.5-month-old MT-FS line 4 transgenic
mouse (bottom) and its littermate control
(top). B, Gross analysis of the testes of a 6-week-old
wild-type mouse (left) and a littermate MT-FS mouse
(right). C, Testis weights of 6-week-old mice. Values
shown are mean ± SEM. The number of mice used is
shown in parentheses as follows: wild-type (15), line 7 (14), line 10
(11), line 5 (11), line 9 (13), and line 4 (3). DG, Histology of
testes from a wild-type adult mouse with normal Leydig
(arrows) cells (high power, D), a 6.5-month-old line 9
transgenic mouse at low (E) and high (F) magnification, and a
6-month-old line 5 transgenic mouse (low power, G). Leydig cell
hyperplasia (asterisks) and tubular degeneration
(arrows or "t") are shown. H, Serum FSH levels of
control and MT-FS adult male mice, the majority of which are at 42
days. Values shown are mean ± SEM. The number of male
mice shown in parentheses used in the analyses are as follows:
wild-type (5), line 7 (5), line 10 (4), line 5 (7), line 9 (5), and
line 4 (2).
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Since follistatin is known to function in the reproductive axis, we
tested the fertility of these transgenic mice by intercrossing them to
wild-type controls for up to 1 yr. Offspring from transgenic lines 7
and 10 showed normal fertility. However, transgenic male mice from
lines 4, 5, and 9 [i.e., lines with highest expression of
the transgene mRNA in the liver and testes (Fig. 3A
)] and transgenic
female mice from lines 4 and 9 demonstrated reproductive defects. All
eight line 4 transgenic female mice failed to have offspring,
demonstrating that line 4 female mice are infertile. Five of nine line
4 male mice were infertile whereas the other four of the line 4 male
mice had two or three litters before they became infertile at
approximately 4 months of age. Male and female mice from line 9 sired
two or three litters before becoming infertile at approximately 4.55
months of age. Additionally, three of eight adult female line 4 mice
died of unknown causes.
To determine the causes of the infertility in these follistatin
transgenic mice, morphological and histological analyses were
performed. Morphological studies showed that the follistatin transgenic
male mice had smaller testes than wild-type controls. Testes from lines
7 and 10 were slightly decreased in size compared with control mice,
whereas testes from line 4 were dramatically smaller compared with
controls (Fig. 4B
). Testes weights from all five transgenic lines of
mice and littermate control mice at 6 weeks of age are shown in Fig. 4C
. Testes weights inversely correlated with the follistatin transgene
mRNA expression in the testes; in lines 4, 9, and 5, which had the
highest expression of the MT-FS transgene mRNA in the testis (Fig. 3A
),
the testes were smaller compared with lines 7 and 10, which had the
lowest mRNA expression. Histological analysis demonstrated Leydig cell
hyperplasia and partial or total tubular degeneration in adult testes
from lines 5 and 9 after 4 months of age. In contrast to the testes of
wild-type mice (Fig. 4D
), spermatogenesis was absent in these tubules,
and only a few Sertoli cells remained (Fig. 4
, EG). Surprisingly,
sections of testes from line 4 show fairly normal stages of
spermatogenesis and normal levels of spermatozoa (data not shown)
despite being infertile. As might be expected and consistent with their
fertility, testes from lines 7 and 10 always show normal stages of
spermatogenesis despite having slightly decreased testis size. To
determine the testis cell type where the transgene was expressed,
in situ hybridization was performed using the SV40
3'-untranslated region (UTR) and poly A probe. The MT-FS transgene mRNA
signal appeared to be present at high levels in both spermatogonia and
Sertoli cells. The signal was not detected in Leydig cells, elongating
spermatids, round spermatids, and late pachytene and diplotene
spermatocytes. Consistent with these findings, endogenous expression of
MT-I mRNA is also expressed in Sertoli cells and spermatogonia in adult
mouse testis (36).
Similar to the males, the transgenic female mice also demonstrated
defects in the reproductive axis. Female mice from line 4 were
infertile, and many of the female mice from line 9 eventually became
infertile at 4 to 5 months of age. Figure 5A
shows the morphological appearance of
the ovaries and uteri of a line 4 female mouse (right) and
its wild-type littermate (left) analyzed at 4.5 months of
age. Compared with the wild-type controls, ovaries from the line 4
female were small, and the uterus was thin, similar to the
GnRH-deficient hypogonadal (hpg) mouse (37, 38).
Histological analysis of these transgenic mice demonstrated small
ovaries and a block in folliculogenesis. Ovaries from a severely
affected 4.5-month-old line 4 transgenic female demonstrated an early
block in folliculogenesis before antral follicle formation (Fig. 5
, C
and D). In the least affected line 4 and 9 transgenic females,
folliculogenesis progressed to the early secondary (antral) follicle
stage (Fig. 5E
); however, there were no tertiary follicles or corpora
lutea present in these ovaries as compared with ovaries from wild-type
mice (Fig. 5B
). In addition, follicular atresia was present (Fig. 5E
),
similar to ovaries from FSH-deficient female mice (39). Ovaries from
another line 4 female mouse demonstrated seminiferous tubule-like
structures in the ovary (Fig. 5
, F and G), similar to the ovaries from
inhibin-deficient mice that have gonadal sex cord-stromal tumors (Refs.
2, 31, and 40; see Discussion). Most of the line 5 female
mice had normal ovaries and were fertile even at later timepoints in
adult development. However, ovaries from some of the line 5 females
demonstrated abnormalities in folliculogenesis with one mouse having a
block in folliculogenesis at the early primary follicle stage. In
situ hybridization analysis of ovaries from line 4 female mice
showed that the transgene mRNA was expressed weakly in stromal
(interstitial) cells (data not shown).

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Figure 5. Morphology and Histology of the Reproductive Tract
and Ovaries of Wild-Type and MT-FS Transgenic Female Mice
A, Gross analysis of the reproductive tract (uterus and ovaries) of a
4.5-month-old wild-type (left) and MT-FS transgenic line
4 mouse (right). BG, Histological analysis of adult
ovaries. B, Wild-type, mouse ovary (low power). Black
arrow, Corpus luteum; white arrow, antral
follicle. C and D, An ovary from the 5-month-old transgenic line 4
mouse shown grossly in panel A at intermediate (C) and high (D) power
magnification, respectively. Note the small ovary and the arrest of
folliculogenesis at the primary follicle stage. Arrows,
Primary follicles. E, An ovary from a 9-month-old transgenic line 9
mouse. An atretic follicle is shown at the left
(white arrow). F and G, An ovary from a 5.5-month-old
female line 4 mouse at intermediate (F) and high (G) power
magnification, respectively. Note the seminiferous tubule-like
structures (S) with cells with nuclei (arrows) that
resemble Sertoli cells.
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Serum and Pituitary Analysis
Activins stimulate pituitary FSH synthesis and secretion in
vitro (1, 2), and activin receptor type II-deficient mice have
suppressed FSH levels (23). Follistatin has been shown to block activin
action at the level of the pituitary in vitro (9). Since
many of the ovary and testis defects in the follistatin transgenic mice
were similar to FSH-deficient (39) and activin receptor type
II-deficient (23) mice, we investigated whether any of the
reproductive defects in our follistatin transgenic mice could be due to
the suppression of FSH by follistatin. In the adult male mice, the FSH
levels, as measured by RIA, were significantly suppressed only in the
line 4 transgenic males compared with control mice (Fig. 4H
). Decreased
FSH levels were also observed in the adult transgenic line 4 females
(24.3 ± 1.9 ng/ml.; n = 5), comparable to the suppressed FSH
levels seen in activin receptor type II knockout mice (35.2 ± 5.2
ng/ml; Ref.23). Consistent with the morphological (i.e.
thin uterus and small ovaries) and histological (i.e. block
in folliculogenesis) findings and suppressed serum FSH, the line 4
female mice had serum estradiol levels below the sensitivity of the
assay (<5 pg/ml). We also measured the serum FSH levels in
gonadectomized adult wild-type and line 5 transgenic males. The results
showed no significant difference in FSH levels between the
gonadectomized MT-FS transgenic male mice (105 ± 13 ng/ml; n
= 4) and nontransgenic controls (107 ± 8 ng/ml; n = 3).
Neither "free" follistatin nor activin A were detected in the serum
of the follistatin transgenic mice (41, 42). These data suggest that
the major effects of overexpression of follistatin were at the level of
the gonads (see Discussion).
The MT-FS transgenic male mice that displayed reproductive defects were
also analyzed to determine whether there were any secondary
abnormalities in steroid production. Since androgen biosynthesis in
males occurs mainly in the interstitial Leydig cells, the serum
testosterone level of transgenic male mice that display Leydig cell
hyperplasia in the testis was determined by RIA. The results showed no
significant difference in serum testosterone levels in these transgenic
mice (1.7 ± 1.1 ng/ml; n = 7) compared with adult wild-type
mice (1.9 ± 1.7 ng/ml; n = 5) consistent with grossly normal
secondary sex organs (i.e. seminal vesicles, prostate). This
suggests that the androgen biosynthesis pathway in these hyperplastic
Leydig cells might be functionally suppressed or abnormal.
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DISCUSSION
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Follistatin is an activin binding protein that has been shown to
antagonize the function of activin in a number of assays (7, 9) and may
also modulate activin action in vivo at the cell surface
(22, 43). In the present study, a "gain of function" mouse model
was generated to further understand the function of follistatin in
mammalian physiology. Despite the expression of the MT-FS transgene
during early embryogenesis, and in contrast to mice lacking follistatin
(22), mice overexpressing the follistatin transgene were viable and
developed to adulthood. The most obvious finding in three of the
follistatin transgenic lines with the highest transgene expression was
shiny, irregular fur (Fig. 4A
). This might have been expected based on
our previous data that follistatin-deficient mice have shiny, taut
skin, whisker defects, and aberrant keratin 6 expression in the
interfollicular epidermis (22), and that activin ßA-deficient mice
lack whiskers (21). Consistent with an important role of activins and
follistatin in the skin, we have recently shown that radiolabeled
activin A binding in the skin of follistatin-deficient embryos at E18.5
was absent compared with wild-type littermates (44). Taken together,
these findings further suggest that follistatin may bind activins and
act as an important reservoir for activins in the epidermis.
In adult rats, activin ßA and follistatin mRNA are expressed in
Sertoli cells surrounding leptotene spermatocytes at stages IXXI, and
activin is a potent stimulator of spermatogonial and Sertoli cell
proliferation (3, 17). Since the role of follistatin in adult
reproduction could not be addressed by our conventional knockout of
follistatin because the mutant mice died soon after birth (22), the
present gain-of-function studies allowed us to ascertain the function
of follistatin in mammalian reproduction. Overexpression of follistatin
resulted in decreased testis size, seminiferous tubular degeneration,
and an arrest of spermatogenesis in MT-FS lines 5 and 9. Although a
similar decrease in testis size was seen in FSH-deficient (39) and
ActRII-deficient mice (23), which have absent and decreased FSH levels,
respectively, these knockout male mice were still fertile.
Interestingly, the serum FSH levels of MT-FS transgenic mice were only
suppressed in line 4 male mice that had the broadest expression of the
transgene. This suggests that the decrease in testes size in the MT-FS
transgenic mice is caused by follistatin acting locally to block
activin, consistent with a hypothetical testis-specific activin
knockout. However, it is unclear why these follistatin overexpressor
mice do not mimic the phenotype of ActRII-deficient mice (23). One
possibility is that activins continue to signal via ActRIIB in the
absence of ActRII. Alternatively, follistatin may regulate the function
of other TGF-ß family members. For example, follistatin-deficient
mice (22) have skeletal defects reminiscent of BMP5-deficient mice, and
high concentrations of follistatin can block the effects of BMP7 and
BMP4 in vitro (45, 46). Adult bone morphogenetic
protein 8b (BMP8b) knockout male mice demonstrate increased apoptosis
of spermatocytes leading to germ cell depletion, tubule degeneration,
and infertility (47). Thus, overexpression of follistatin may also
block BMP8b to cause the infertility, and under physiological
conditions, follistatin may normally play a role in maintenance of
spermatogenesis by modulating the function of BMP8b in the mouse. In
addition, Leydig cell hyperplasia was present in the testis of line 5
and 9 male mice. In Müllerian inhibiting substance (MIS, a member
of TGF-ß family) knockout male mice or MIS/inhibin double-mutant male
mice, Leydig cell hyperplasia and Leydig cell neoplasia, respectively,
were observed (48, 49). Some aspects of the Leydig cell hyperplasia in
our follistatin transgenic mice may be due to antagonism of MIS
(i.e. follistatin overexpression may act functionally like
an MIS null mutation).
Activin stimulates granulosa cell proliferation and follicular
development in vitro (3, 16). Varying levels of infertility
were observed in line 4 and 9 female mice that demonstrated thin uteri,
small ovaries, and a block in folliculogenesis. Similar to the ovarian
findings in mice lacking FSH [i.e. only primary follicles
were observed (39)], folliculogenesis in many of the MT-FS transgenic
female mice from these two lines is halted between the primary and
secondary follicle stages, follicular atresia was present, and in most
ovaries, no corpora lutea were seen. Interestingly, one line 4 female
mouse exhibited Sertoli tubule-like structures (Fig. 5
, F and G),
similar to ovarian tumors from inhibin-deficient mice and
inhibin-deficient ovaries transferred to the bursa of wild-type control
mice (2, 31, 40). Since FSH levels were only suppressed in line 4
female mice, a local role of follistatin in the ovary may also be
responsible for the ovarian defects by blocking activins and/or other
TGF-ß family members. Mice deficient in the oocyte-specific protein
growth differentiation factor 9 (GDF-9), a member of the TGF-ß
family, demonstrate a block in folliculogenesis at the one-layer
primary follicle stage (50). High levels of follistatin in our MT-FS
transgenic mice may bind to GDF-9 and block its effects, thereby
resulting in folliculogenesis defects similar to the GDF-9-deficient
mice. Thus, if follistatin interacts with other members of the TGF-ß
superfamily, this would suggest further promiscuity in the TGF-ß
superfamily signaling pathways and give us important insight into human
infertility and potential treatments.
We have shown that activin signaling through activin receptor type II
directly causes the cancer cachexia-like syndrome in inhibin-deficient
mice (27, 28). Other studies have demonstrated that activin inhibits
hepatocyte proliferation and that follistatin accelerates liver
regeneration (24, 25, 26, 29, 30, 51). Since the MT-FS transgene is highly
expressed in the liver of lines 4 and 5, we will breed these mice to
our inhibin-deficient mice to determine whether overexpression of
follistatin can block this activin-mediated cancer cachexia-like
syndrome. These future studies will be important to further understand
the roles of follistatin in vivo.
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MATERIALS AND METHODS
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Construction of the MT-FS Transgene
A 1.8-kb mouse metallothionein-I promoter fragment (32, 33) was
placed upstream of the six coding exons of the mouse follistatin gene
(5.1 kb, Refs. 2 and 20; Fig. 1
). To distinguish the follistatin
transgene mRNA from the endogenous follistatin mRNA, a 973-bp
BamHI/ClaI fragment, which contains the simian
virus 40 (SV40) untranslated (UTR) and polyA sequences, was placed in
the 3'-untranslated region of the transgene. The plasmid containing the
MT-FS transgene was digested with EcoRI and BamHI
to release the transgene fragment, which was subsequently isolated
using GENECLEAN (BIO 101, La Jolla, CA).
Generation of Transgenic Mice and Morphological and Histological
Analysis
The MT-FS transgene DNA (a 7.9-kb linearized fragment) was
microinjected into the pronucleus of fertilized eggs generated from
C57BL/6/C3H x ICR hybrids (52). Microinjected eggs were
transferred to the oviducts of foster mothers (ICR female mice, 67
weeks old). Twenty four mice were obtained from six foster mothers.
Genomic (tail) DNA (
5 µg) from the founder mice and offspring was
digested with XbaI, electrophoresed on 0.7% agarose gels,
and subjected to Southern blot analysis using the SV40 3'- UTR and
polyA probe as described above. All transgenic founder and F1 mice were
intercrossed with wild-type or transgenic littermates beginning at 6
weeks of age. Fourteen, eight, and six breeding pairs of transgenic
male or female mice from lines 4, 9, and 5, respectively, were set up
for up to 1 yr to test the fertility of these mice. The number of
offspring born and frequency of births were recorded. Transgenic mice
and littermate controls were weighed weekly on the same day until 26
weeks of age. Ten pairs of testes from all five transgenic lines of
mice and 11 pairs of testes from control mice were weighed at 6 weeks
of age, and serum from these mice were also collected at the same time.
Any mice that demonstrated infertility for
3 months were
analyzed morphologically and histologically. Testes were fixed in
Bouins solution overnight and then transferred to 70% ethanol in
saturated lithium carbonate, and rinsed daily for approximately 1 week.
Other tissues were dissected and immediately fixed by immersion
overnight in 10% neutral buffered formalin. The tissues were embedded
in paraffin, and 5-µm sections were cut. Sections from ovaries and
testes were stained with hematoxylin and periodic acid Schiff stain
reaction. Sections from other tissues were stained with hematoxylin and
eosin.
Serum and Pituitary Analysis
Serum and pituitary levels of LH and FSH in wild-type and MT-FS
transgenic mice were analyzed by RIA using kits obtained from the
National Hormone and Pituitary Distribution Program, National
Institutes of Diabetes, and Digestive and Kidney Diseases as described
previously by our group (23, 31, 35, 39, 50). Serum levels of
testosterone and estradiol from control and transgenic mice were
determined using Diagnostics Systems Laboratories, Inc. (Webster, TX)
RIA assay reagents as previously described (2). Serum activin levels
were determined by enzyme-linked immunosorbent assay (ELISA) (Serotec,
Ltd., Raleigh, NC) as described (42). Serum collected from
inhibin-deficient mice was used to validate the mouse activin assay.
Native mouse activin A was diluted linearly and in parallel to the
recombinant human activin A standard curve. All samples were run within
one assay. The intraassay coefficient of variance for positively
detected samples was 5.2%. All samples from PMSG-induced mice, control
wild-type mice, and MT-FS transgenic mice were below the detection
limit of the assay (0.078 ng/ml). Free follistatin was measured in a
two-site ELISA assay format as described (41). The assay detects
recombinant human follistatin 288; however, activin A interferes with
the detection of follistatin in this assay. Follistatin was not
immunodetected using this assay format in the serum of follistatin
transgenic mice. The assay is sensitive and specific for human
follistatin but may not detect mouse follistatin. This possibility
cannot be tested without a mouse follistatin standard.
RNA Analysis
Total RNA was extracted from fresh tissues from both transgenic
and wild-type mice using RNA STAT-60 reagent (Leedo Medical
Laboratories, Houston, TX) according to the manufacturers protocol.
For Northern blot analysis, 15 µg of total RNA were loaded on a 7.6%
formaldehyde-1.2% agarose gel, and the RNA was transferred to Hybond-N
(Amersham, Arlington Heights, IL) nylon membrane as described (50). The
membrane was hybridized with the same SV40 UTR/poly A probe used for
Southern blot analysis as described (50). For the quantitative control,
the membrane was stripped and rehybridized with a mouse 18S ribosomal
RNA cDNA probe. To determine whether the MT-FS transgene was expressed
at early embryonic stages, total RNA from E6.5 or E7.5 embryos from
timed matings of carrier transgenics was processed using the RNA
STAT-60 reagent, and 10% of the total RNA was used for RT-PCR
analysis. RT-PCR was performed using the Titan One Tube RT-PCR system
(Boehringer Mannheim, Indianapolis, IN) according to the
manufacturers protocol.
In Situ Hybridization Analysis
In situ hybridization was performed as described by
Albrecht et al. (53). In brief, freshly dissected mouse
testes and ovaries were fixed in Bouins and freshly prepared 4%
paraformaldehyde-PBS, respectively. Testes samples were immersed for
3 h in Bouins and then transferred to 70% ethanol. After
fixation, the tissues were paraffin embedded and sectioned at 5 µm.
The SV40 3'-UTR and polyA riboprobe, labeled with
[
-35S]UTP, was used to detect the follistatin
transgene expression. Hybridization was carried out at 5055 C with
5 x 106 cpm/slide riboprobe for 16 h in 50%
deionized formamide, 0.3 M NaCl, 20 mM Tris HCl
(pH 8.0), 5 mM EDTA, 10 mM NaPO4
(pH 8.0), 10% Dextran sulfate, 1x Denhardts reagent, and 0.5
µg/ml yeast RNA. A high-stringency wash was carried out in 0.1x
sodium chloride-sodium citrate at 65 C. Dehydrated sections were
exposed to X-OMAT film for 13 days. After film exposure, slides were
dipped in NTB-2 Kodak emulsion and exposed for 110 days at 4 C. After
the slides were developed and fixed, they were stained with hematoxylin
or hematoxylin/eosin and mounted with permount for photography.
Sperm Parameters
Epididymides were dissected from 6- to 7-week-old male mice, and
the sperm were allowed to disperse into M-2 medium upon incubation at
37 C for 1530 min. A 1:10 dilution was used for hemocytometric counts
and for motility testing, while a 1:20 dilution was used for viability
testing using Eosin-Y (52, 54).
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. Grace Hamilton and Mr. Bliss Walker for
histological technical assistance, Ms. Julia Elvin for critical review
of the manuscript, and Ms. Shirley Baker for help in manuscript
preparation. We thank Dr. Debra Wolgemuth for interpretative analysis
of in situ hybridization results on the transgenic testes.
We thank Dr. Richard Palmiter for the gift of the metallothionein I
promoter and Dr. Patrick Sluss for the follistatin antibodies.
These studies were supported by NIH Grants HD-32067 and CA-60651 (to
M.M.M) and Reproductive Center Grant HD-07495.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin M. Matzuk M.D., Ph.D., Departments of Pathology, Cell Biology, and Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
Received for publication May 30, 1997.
Revision received October 2, 1997.
Accepted for publication October 20, 1997.
 |
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