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-Subunit Gene
Department of Biochemistry, Molecular Biology, and Cell Biology, and Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Kelly E. Mayo, Ph.D., Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208. E-mail: k-mayo{at}northwestern.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit gene were
generated. A transgene that includes the mouse metallothionein-I gene
promoter (MT-) fused to the rat inhibin -subunit precursor coding
sequences was used to produce three lines of transgenic mice. Transgene
mRNA is expressed in numerous tissues, including the pituitary, liver,
testis, ovary, and kidney. Inhibin -subunit protein was also
increased in transgenic pituitary and ovary. Serum inhibin -subunit
levels are highly increased compared with control mice. Inhibin
ßA- and ßB-subunit protein amounts are
lower in transgenic ovaries compared with wild type, although serum
levels of activin A are not significantly reduced in transgenic female
mice. FSH levels are reduced in both male and female transgenic mice,
whereas LH levels are increased in MT- female mice. MT-
transgenic females are subfertile and exhibit a 52% reduction in
litter size compared with wild-type females. The smaller litter size of
MT- female mice was correlated with a reduction in the number of
oocytes ovulated during a normal cycle. Treatment of the transgenic
females with exogenous gonadotropins resulted in an ovulation rate
similar to that of stimulated wild-type animals, suggesting that
altered gonadotropin levels may be responsible for the decreased
ovulation rates. MT- transgenic male mice are fertile and sire
litters of equivalent size to those sired by wild-type males, despite
an approximately 50% reduction in sperm numbers. These results
indicate that overexpression of the rat inhibin -subunit gene in
mice leads to a disruption of the normal inhibin-to-activin ratio and
to reproductive deficiencies, and they support the hypothesis that
inhibin and activin act to regulate FSH secretion in
vivo and are essential for normal gonadal function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and one of two
related ß-subunits (ßA and
ßB) (1, 2, 3, 4), whereas activin is a
dimer of two of the ß-subunits (5, 6). The subunit RNAs
are individually expressed at varying levels in tissues, including the
ovary, testis, adrenal, pituitary, brain, placenta, and spleen
(7). Inhibin -subunit mRNA is primarily expressed in
the granulosa cells of the ovary (8), the Sertoli cells of
the testis (9), and the adrenal cortex (10).
Inhibin and activin were identified for their ability to inhibit and
stimulate, respectively, FSH secretion and synthesis from cultured
anterior pituitary cells (1, 2, 3, 4, 5, 6, 11). The inhibins and/or
activins have also been shown to regulate diverse physiological
functions, including early embryonic development (12, 13, 14),
erythroid differentiation (15), pituitary GH biosynthesis
(16), hypothalamic oxytocin secretion (17),
and neuronal survival (18). Inhibin production is tightly regulated throughout the female reproductive cycle. On the afternoon of proestrus of the rodent estrus cycle, just before the preovulatory FSH and LH surges, the granulosa cells of developing follicles produce high levels of inhibin A (8, 19). After the gonadotropin surges, ovarian inhibin production declines (20). Low inhibin levels persist through the morning of estrus, providing an environment permissive to increased FSH and the generation of the secondary FSH surge. Treatment of proestrus rats with charcoal-extracted follicular fluid as a source of inhibin prevents the secondary FSH surge (21). The secondary FSH surge is responsible for the recruitment of new follicles into the antral pool (22, 23), and FSH stimulates inhibin expression in newly recruited follicles (19, 24). Thus, appropriate inhibin levels are important to maintain the estrus cycle and for normal follicle development.
Mice deficient in inhibin after the disruption of the inhibin
-subunit gene illustrate the in vivo importance of
inhibin for normal gonadal function. Inhibin -subunit-deficient mice
develop ovarian and testicular stromal tumors (25), and if
gonadectomized before gonadal tumorigenesis, they develop adrenal
tumors (26). Maturing oocytes and spermatozoa are
initially present in the gonads of inhibin -subunit-deficient mice,
although the development of gonadal stromal tumors leads to the arrest
of gametogenesis (25). These animals die prematurely at
3–4 months of age and thus do not permit an extensive analysis of
inhibin in reproductive function in the adult. An alternative approach
for establishing the importance of inhibin and its appropriate
regulation in the reproductive axis is to investigate phenotypes that
arise as a consequence of overexpression of the inhibin -subunit in
transgenic mice. To determine the effects of inhibin misregulation on
activin expression, on FSH production, and on gonadal function, we
generated transgenic mice that overexpress the rat inhibin -subunit
cDNA. This paper reports on the reproductive phenotypes of these mice,
and the accompanying paper describes gonadal pathologies related to
inhibin -subunit overexpression in transgenic mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA was cloned into the vector pEV-142
(provided by Dr. Richard Palmiter, University of Washington, Seattle),
which includes a mouse metallothionein-I promoter (MT-) and a human
GH RNA processing and polyadenylation site (27, 28, 29). The
vector was digested with EcoRI to linearize the transgene,
and the linear DNA was subsequently isolated from an agarose gel.
Transgenic animals were produced at the Northwestern University-Markey
Developmental Biology Center core facility under the direction of Dr.
Phillip Iannoccone. Transgene DNA at a concentration of 1–10 ng/µl
was injected into the pronucleus of CD-1 (Swiss Webster albino outbred
mice, Charles River Laboratories, Inc., Wilmington, MA)
zygotes. Microinjected eggs were transferred to the oviducts of
pseudopregnant foster mothers. Genomic DNA was isolated from tail
biopsies of the 11 potential founder mice born. DNA (20 µg) was
digested with KpnI, fractionated on a 0.7% agarose gel, and
hybridized with the 1.4-kb inhibin cDNA probe. Three founder male
mice were identified and used to establish three separate transgenic
lines (lines MT- A–C). Transgene copy number was determined by
Southern blot analysis with 10 µg of DNA prepared as described
(29). Founder males were crossed with CD-1 females, and
all subsequent generations were raised in a room with a controlled
photoperiod (14 h of light, 10 h of dark) and temperature (22–25
C). All lines stably transmit the transgene at the expected 50%
Mendelian frequency. Animals were tested for the presence of the
transgene by dot blot analysis of 10 µg of genomic (tail) DNA using
the 1.4-kb rat inhibin cDNA as a hybridization probe.
Transgene RNA analysis
RNA used for RT-PCR was isolated by homogenization of tissues in
guanidinium thiocyanate and ultracentrifugation through a CsCl cushion
(30). Total RNA yield was estimated by
A260. RNA (5 µg) was reverse transcribed at 42
C using random hexamer primers and avian myeloblastosis virus
reverse transcriptase (Promega Corp., Madison, WI) in a
total volume of 20 µl. A separate negative control RT reaction was
performed with 5 µg of RNA from each sample (excluding avian mosaic
virus reverse transcriptase) to ensure the absence of genomic DNA
contamination. The PCR reaction mix included 5 µl of the RT reaction,
[-32P]dCTP (2 µCi at 3000 Ci/mmol),
Taq DNA polymerase (Perkin-Elmer Corp.,
Norwalk, CT), and 70 ng of each oligonucleotide primer. The
oligonucleotide primers used to detect the MT- transgene included
MT-I (5'-CCA CGA CTT CAA CGT CCT GAG-3') and MI-3 (5'-CCT GTA CCA AGG
ACA CAG GC-3'), which amplified a 580-bp transgene mRNA product. In a
separate PCR reaction, the oligonucleotide primers L19-A (5'-CTG AAG
GTC AAA GGG AAT GTG-3') and L19-B (5'-GGA CAG AGT CTT GAT GAT CTC-3')
were used to amplify a 194-bp region of ribosomal protein L19 as an
internal control (31). Amplification was carried out for
24 cycles using an annealing temperature of 58 C in a
Perkin-Elmer Corp. thermal cycler. The PCR products were
separated by size using electrophoresis on 6% polyacrylamide gels and
exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY).
In situ hybridization
Ovaries were removed from storage at -80 C and brought to -20
C in a Reichert 820 cryostat (Buffalo, NY). Sections were cut at 20
µm and mounted onto VWR Scientific Superfrost Plus slides (West
Chester, PA) for in situ hybridization. Ovarian sections
were fixed in 5% paraformaldehyde (pH 7.8) for 5 min, rinsed in 2x
standard sodium citrate (SSC) for 5 min, washed in 0.25% acetic
anhydride in 0.1 M triethanolamine (pH 8.0) for
10 min, and then dipped in 2x SSC. Sections were dehydrated through an
ethanol series and vacuum dried until hybridization. Antisense and
sense [35S]UTP-labeled RNA probes were
synthesized using T7 or SP6 polymerase. The probe (1.1 x
107 cpm/ml with a specific activity of
3.0–6.0 x 107 cpm/µg) in hybridization
buffer [50% formamide, 0.3 M NaCl, 10
mM Tris (pH 8.0), 1 mM
EDTA, 1x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, 10
mM dithiothreitol, 600 µg/ml yeast tRNA, and
600 µg/ml poly(A)] was applied to the tissue sections, and the
sections were overlaid with a coverslip. Slides were hybridized in a
humidified incubator at 47 C for 16–20 h. After hybridization, the
coverslips were removed in 4x SSC, and sections were treated with 20
µg/ml RNase-A at 37 C for 1 h, washed in decreasing
concentrations of SSC down to 0.1x SSC at 65 C, and dehydrated in an
ethanol series. The slides were exposed to Kodak Biomax MR
film for 3–5 d and then processed for liquid emulsion autoradiography
using Kodak NTB-2 emulsion. Slides were developed after 2
wk and stained with hematoxylin.
Protein blot analysis
Tissues were removed and immediately frozen on dry ice. Tissues
were ground by mortar and pestle and collected in 40 µl of protein
extraction solution [10 mM Tris (pH 7.5), 0.5
M NaCl, 1 mM MgCl2, 0.1%
Triton X-100, and one complete mini protease inhibitor cocktail tablet
per 10 ml (Roche, Indianapolis, IN)]. Homogenates were
exposed to two freeze-thaw cycles (dry ice, +37 C) and briefly
centrifuged, and the supernatant was transferred. Protein concentration
was determined by a bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) using 1-µl dilutions of protein on
microtiter plates. Protein (60 µg) was equally loaded into each lane
for each tissue. Protein was mixed with 4x NuPAGE LDS
(Novex, Carlsbad, CA) at a final concentration of 0.1
M dithiothreitol and heated at 70 C for 10 min before
loading. Proteins were electrophoresed in NuPAGE 4–12% Bis-Tris gels
(Novex), immersed in 1x MES buffer [20x = 1
M 2-(N-morpholino)-ethanesulfonic acid, 1
M Tris Base, 70 mM SDS,
20.5 mM EDTA; 200 µl of NuPAGE antioxidant
between gels in each apparatus; Novex, Carlsbad, CA], and
subsequently transferred in 1x transfer buffer (10x = 0.25
M Tris Base, 0.5 M glycine)
mixed with 20% methanol. Blots were incubated overnight with shaking
in 10% milk-Tris-buffered saline (TBS)-Tween [20
mM Tris (pH 7.5), 500 mM
NaCl, 0.1% Tween] blocking solution at 4 C. Blots were incubated with
, ßA, or ßB primary
antibodies at the following concentrations in 5% milk-TBS-Tween:
(1:2000) for 1 h [anti- 1–26 GY affinity purified],
ßA (1:1000) for 4 h
[anti-ßA 81–113 affinity purified], and
ßB (1:1000) for 4 h
[anti-ßB 80–112 affinity purified].
Antibodies were provided by Dr. Wylie Vale (Salk Institute, San Diego,
CA). Blots were washed in milk-TBS-Tween and incubated with secondary
antirabbit Ig horseradish peroxidase-linked whole antibody (from
donkey) (Amersham Pharmacia Biotech, Little Chalfont,
Buckinghamshire, UK) (1:10,000) in 5% milk-TBS-Tween for 30 min.
Secondary antibody-specific signal was detected with an ECL kit
(Amersham Pharmacia Biotech) using an overnight
exposure.
Hormone measurements
All intact female mice were cycled before collection of
serum for hormone analysis. Estrous cycle stages were determined by
daily examination of vaginal cytology. Those animals demonstrating a
minimum of two consecutive 4- to 5-d cycles were killed on the morning
of metestrus or diestrus. Gonadectomized animals were maintained for 1
wk before serum collection. To examine potential alterations in hormone
levels based on age, measurements were performed on mice ranging from 3
to 12 months in age. Serum hormone measurements were determined by RIA
at the Northwestern University P30 Center RIA Core Facility under the
direction of Drs. John Levine and Neena Schwartz. National Institute of
Diabetes and Digestive and Kidney Diseases antiserum and standards
(rLH-RP-3 standard/rLH-S-11 antibody and rFSH-RP-2 standard/rFSH-S-11
antibody) were used for LH and FSH measurements. FSH and LH results are
expressed as nanograms per milliliter. FSH assay sensitivity was 0.05
ng/sample or 1.0 ng/ml, and LH assay sensitivity was 0.01 ng/sample or
0.2 ng/ml. The interassay coefficients of variation were 7.9% and
17.5% for female serum FSH and LH measurements, respectively. The
interassay coefficients of variation were 15.8% and 9.9% for male
serum FSH and LH measurements, respectively. Reagents provided by Dr.
Wylie Vale (Tyr 27 rat inhibin 1–27 standard/sheep anti-Tyr 27 rat
inhibin 1–27 antibody 795) were used for the inhibin -chain
assay as described (32, 33). The RIA measures total
inhibin (free and dimeric inhibin) (32, 33), and
inhibin results are expressed as picomoles per milliliter. The
interassay coefficient of variation for all inhibin measurements was
9.3%. We attempted to measure serum dimeric inhibin levels using the
human inhibin A dimer ELISA (Serotec, Oxford, UK);
however, sera from MT- or wild-type littermate mice were nonparallel
in the assay. Sera from other mouse strains such as C57BL16 were
parallel in the assay, suggesting that serum from the CD-1 mouse strain
specifically interferes with this human inhibin A dimeric ELISA. Serum
dimeric activin A levels were measured with the human activin A dimer
ELISA (Serotec) according to the manufacturer’s protocol
with undiluted serum.
Fertility measurements
MT- (transgenic) mice and control (nontransgenic littermate)
mice were paired with wild-type (CD-1; Charles River Laboratories, Inc.) mice of the opposite sex. Each mating pair
was housed together until three to eight litters of offspring were
produced. The litter sizes of each mating pair were averaged (lines A
and C), and the time interval between births was recorded (line A).
Female age varied from 3–6 months at the beginning of the study to
7–11 months by the end. Wild-type or transgenic females in this age
range (3–11 months) did not display significant variance in litter
size as a result of age. Males used in this study ranged from 3 to 10
months of age.
Ovulation analysis
Female MT- (transgenic) mice and female control
(nontransgenic littermate) mice were housed with wild-type (CD-1;
Charles River Laboratories, Inc.) vasectomized males.
Females were examined every morning for the presence of a copulatory
plug. Those females with copulatory plugs were killed at midday, and
the oviducts were removed and transferred into room temperature M2
medium (Sigma, St. Louis, MO). Oviducts were flushed and
opened to release the ova/cumulus cell complexes. After removal of the
cumulus cells with hyaluronidase, the ova were counted.
Gonadotropin-stimulated females were injected with 5 IU of PMSG
followed by 3 IU of hCG 45 h later. After the hCG injection,
females were housed and ova were recovered under the same conditions
described above. Those females with copulatory plugs were killed 20–22
h after hCG treatment.
Sperm parameters
The epididymis was dissected from the scrotum of 2- to
3-month-old adult male mice. The sperm were squeezed from the
epididymis with watchmaker’s forceps. The sperm were incubated in 2 ml
of M2 medium (Sigma) at 37 C for 15–30 min, diluted 1:10,
counted, and examined for motility (based on flagellar movement) on a
hemocytometer as described (29, 34).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit (27) fused
to the rat inhibin -subunit cDNA was used to generate transgenic
mice that overexpress the rat inhibin -subunit gene product (Fig. 1A
). Southern blot analysis with 10 µg
of genomic DNA isolated from the F0 offspring
identified three transgenic founder males (Fig. 1B) that were bred to
establish three separate MT- transgenic lines. The
KpnI-digested genomic DNA resulted in an approximately
2.0-kb band and a 0.7-kb band when probed with the 1.4-kb rat inhibin
-subunit cDNA (Fig. 1A). Lanes 1, 3, and 6 represent MT-
transgenic lines C, A, and B, respectively (Fig. 1B). The pattern of
hybridization indicates that multiple copies of the transgene are
inserted in tandem. Copy number was determined by comparing 10 µg of
genomic DNA with a series of copy number controls using dot blot
analysis (data not shown) (29). Line A has approximately
12 copies of the transgene (per diploid genome), line B has
approximately 2 copies, and line C has approximately 20 copies.
Southern blot analysis of genomic DNA isolated from offspring indicates
that the transgene is passed to 50% of the offspring, as expected for
all three lines. MT- transgenic mouse lines A and C were used for
all of the studies reported here.
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coding sequence (Fig. 2A). Transgene mRNA expression was found
in all transgenic tissues examined, including the ovary, testis,
pituitary, kidney, and liver of line A and line C MT- transgenic
mice (Fig. 2B). Although the RT-PCR is not quantitative, based on
Northern analysis, transgene mRNA levels are particularly high in the
pituitary (data not shown). As expected, no transgene mRNA expression
was observed in wild-type littermates. In situ hybridization
of MT- and wild-type mouse ovaries revealed that inhibin expression
is detected in the granulosa cells of maturing follicles, as expected
(Fig. 3, left). Additional
inhibin -subunit gene expression is observed in the interstitial and
stromal cells of MT- transgenic ovaries, indicating increased levels
of inhibin -subunit gene mRNA expression in these cell types of the
transgenic ovary compared with controls. Inhibin
ßA-subunit expression is localized to the
granulosa cells of mature antral follicles in both MT- transgenic
and wild-type control ovaries, and there do not appear to be
substantial changes in ßA mRNA levels in the
MT- ovaries (Fig. 3, right).
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ßA, and ßB) in
transgenic tissues, Western analysis was performed with antibodies
specific for the individual subunit proteins. The Western blots shown
in Fig. 4 are representative of results
from three different experiments. An abundance of mature -subunit
protein (18 kDa) was present in MT- transgenic ovary and pituitary
(Fig. 4A). The protein levels in the transgenic pituitary are
consistent with the Northern and RT-PCR RNA expression studies. No
-subunit protein was observed in the wild-type ovary in the blot
shown; however, with increased incubation time of primary antibody,
-subunit protein was observed in wild-type ovary (data not shown).
Mature ßA-subunit protein (14 kDa) was detected
in the ovary of wild-type mice, and levels were slightly reduced in the
MT- ovary (Fig. 4B). Very small amounts of
ßB-subunit protein (14 kDa) were also detected
in the ovary of wild-type mice, and this level was reduced in the ovary
of MT- transgenic mice (Fig. 4C). No mature inhibin/activin subunit
proteins were detected in the negative control tissue (liver), although
a larger molecular mass band that might represent the precursor protein
was observed.
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-chain (measures free -subunit and dimeric inhibin) was
performed on sera samples collected from MT- transgenic and control
mice (32, 33). Serum total inhibin levels were
significantly increased in MT- transgenic male and female mice
compared with wild-type mice (Fig. 5, A
and B). After ovariectomy, there was a significant reduction in serum
total inhibin in wild-type female mice. In contrast, total inhibin
levels remained increased in the MT- female mice, suggesting
nongonadal expression of the transgene. This is consistent with the
RT-PCR results, which show transgene expression in numerous nongonadal
tissues. Basal FSH levels (Fig. 6, A and
B) were significantly reduced in the MT- mice compared with
wild-type controls, consistent with the idea that biologically active
inhibin is produced that is able to suppress pituitary FSH synthesis or
secretion. In addition, FSH levels after gonadectomy were significantly
repressed in MT- female mice compared with controls (wild type,
24 ± 1.2 ng/ml; line A, 15.9 ± 1.2 ng/ml; line C, 17.4
± 1.0 ng/ml; P < 0.01). Serum FSH levels were
measured for female mice ranging from 3 to 12 months in age, and
reduced FSH levels remained constant in transgenic females despite age,
whereas FSH levels increased slightly in older wild-type females (data
not shown). In contrast to serum FSH levels, MT- female mice showed
a significant increase in serum LH levels (Fig. 6C), and serum LH
levels increased with advancing age. In contrast to MT- female mice,
MT- male mice displayed a decrease in serum LH, although this was
not significant in either line (Fig. 6D). Dimeric activin A levels were
similar among young transgenic and wild-type female mice (Table 1), but activin A levels decreased
slightly in older transgenic female mice.
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transgenic mice from lines A and C were mated
with wild-type animals. Successive matings of MT- female mice with
wild-type male mice revealed a 52% decrease in litter size for MT-
female mice compared with litters produced by control (nontransgenic
littermate) female mice (Fig. 7). Line A
MT- transgenic females gave birth to an average of 6.5 ± 0.6
pups, line C MT- transgenic females had an average litter size of
5.0 ± 0.3 pups, and nontransgenic female littermates had an
average litter size of 11.5 ± 0.6 pups (P <
0.001). The time interval between births was also slightly greater in
MT- transgenic female mice, with an average of 29.5 d between
births, compared with 25.7 d between births for nontransgenic
littermates.
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transgenic males were mated to control females. MT- males sired
litters (line A, 13.3 ± 0.8 pups; line C, 12.0 ± 0.2 pups)
of comparable size to litters sired by nontransgenic males (11.6
± 0.6 pups) (Fig. 7
). Although MT- male fertility does not appear
to be impaired, MT- males have reduced sperm production. Analysis of
sperm parameters showed epididymal sperm numbers to be decreased by
approximately 55–60% in MT- transgenic males compared with
nontransgenic males (Table 2). However,
there was no significant difference in sperm motility, as measured by
the percentage of sperm showing flagellar movement (Table 2).
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transgenic ovaries revealed fewer antral
follicles and corpora lutea than wild-type ovaries, as described in the
accompanying paper, suggesting a follicular defect and/or a potential
decrease in ovulation rates. To test this, the number of oocytes
released from a natural ovulatory cycle was determined for MT-
transgenic female mice and control female mice. The number of oocytes
recovered from MT- transgenic female mice was 54% the number of
oocytes recovered from control female mice. MT- transgenic females
have an average ovulation rate of 8.2 ± 0.9 oocytes for line A
and 8.0 ± 1.8 oocytes for line C, whereas control female mice
have an average of 15.1 ± 0.2 oocytes per ovulation
(P < 0.001) (Fig. 8A).
Thus, the reduction in ovulation rate for transgenic female mice is
similar to the approximately 50% reduction in litter size (Fig.
7).
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transgenic females could be
secondary to abnormal gonadotropin levels. To determine if transgenic
females were capable of responding to exogenous gonadotropins to
increase ovulation rate, MT- transgenic females and control females
were treated with PMSG and hCG. After superovulation, transgenic
females ovulated an approximately equal number of oocytes (line A,
50.5 ± 5.8; line C, 34.5 ± 4.4) as control females
(46.3 ± 5.7) (Fig. 8B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibin transgenic mice. The mouse metallothionein
promoter was used to produce broad expression of the rat inhibin
-subunit cDNA in transgenic mice. Of the 11 mice produced after
pronuclear injection, 3 MT- transgenic founder male mice were
identified (Fig. 1B), thus permitting the establishment of MT-
transgenic lines A, B, and C.
In the adult rodent, inhibin -subunit gene expression is both
spatially and temporally regulated (7, 8). The inhibin
-subunit is primarily expressed in the ovary, testis, adrenal, and
pituitary, whereas the tissue-specific expression patterns of the
inhibin ß-subunits, ßA and
ßB, are less restricted (7). The
metallothionein-I gene is expressed in almost all tissues except thymus
(35). This promoter region of mouse metallothionein-I was
originally used to generate transgenic MT-human-GH mice
(28) and transgene expression in these mice was observed
in many different tissues (35). Similarly, in our MT-
transgenic mice, the expression of the MT- transgene was achieved in
all tissues examined (Fig. 2B). The expression of the MT- transgenic
rat inhibin -subunit in a broad range of tissues, in addition to
endogenous mouse inhibin -subunit expression, should permit the
increased production and assembly of dimeric inhibin in those tissues
that also express the ß-subunits. The suppression of serum FSH in
MT- intact and MT- gonadectomized mice suggests that increased
bioactive dimeric inhibin production was achieved in vivo
(Fig. 6, A and B). Ovariectomized wild-type mice exhibited very low
inhibin levels, whereas serum inhibin levels for ovariectomized
MT- female mice remained increased, indicating nongonadal expression
of the MT- transgene (Fig. 5A). These data support the RT-PCR
findings that demonstrate expression of the transgene mRNA in
nongonadal tissues.
An alternative explanation for the reduced FSH levels in these mice is
that dimerization of the excess free -subunit with endogenous
ß-subunits, or effects of -subunit expression on ß-subunit
synthesis, might reduce the levels of activin dimers and thus adversely
affect activin actions. Consistent with this possibility, we observed
small reductions in the levels of ß-subunit proteins in transgenic
mouse ovaries, and serum levels of activin A were slightly reduced
compared with control mice, although this change does not reach
statistical significance. It may be that both increased inhibin
production and decreased activin production contribute to an altered
inhibin-to-activin ratio and to the observed hormonal and reproductive
phenotypes in MT- transgenic mice.
MT- transgenic mice provide a useful in vivo model for
examining the effects of inhibin on the hypothalamic-pituitary-gonadal
axis. The gonadotropin ratio is significantly altered in MT-
transgenic female mice. FSH levels are repressed in both male and
female MT- transgenic mice, which supports the roles of inhibin and
activin as important negative regulators of FSH secretion in
vivo. The treatment of anterior pituitary cell cultures with
inhibin results in a significant reduction in FSH production
(1, 2, 3, 4). In vivo studies that support the role
of inhibin as a negative regulator of FSH include an early experiment
performed with proestrus rats treated with porcine follicular fluid as
a source of inhibin. After treatment, these animals exhibited
suppression of the secondary FSH surge (21). Additionally,
the infusion of female rats with an inhibin polyclonal antiserum caused
an increase in plasma FSH, thus supporting the importance of inhibin
negative feedback in FSH regulation (36).
It is perhaps surprising that MT- transgenic mice are fertile and
that FSH secretion is not further reduced in these animals. In
vivo, the ability of inhibin to suppress FSH may be dependent on
additional factors. An in vivo study was performed with
cycling female rats treated with recombinant human inhibin A at a
dosage in excess of available circulating inhibin (37).
Rats were treated with inhibin during proestrus, estrus, metestrus, or
diestrus, and serum FSH was measured each hour after treatment. Animals
in proestrus demonstrated a reduction in FSH, although generally no
more than 2-fold. Rats in estrus, metestrus, and diestrus treated with
recombinant inhibin showed no reductions in serum FSH, suggesting that
in vivo regulation of FSH production by inhibin is limited
and cycle dependent (37). These in vivo data
suggest that inhibin may not act alone but instead likely acts with
other factors, such as E, in mediating the down-regulation of FSH
production in a cycle-dependent manner. In vivo studies
performed with E and inhibin further support the coordinated actions of
these hormones in mediating FSH suppression (38).
Ovariectomized rats treated with charcoal-extracted porcine follicular
fluid as a source of inhibin, as well as rats treated with E, exhibited
a decrease in serum FSH (38). Rats treated with both
follicular fluid (inhibin) and E exhibited an added suppression in
serum FSH compared with either treatment alone (38),
suggesting that inhibin and E act in a synergistic manner to reduce FSH
secretion in vivo. E levels are reduced in MT- female
mice, as described in the accompanying paper. This lack of adequate E
negative feedback could limit the suppression of FSH by inhibin in
MT- transgenic female mice.
The basis for increased LH levels in MT- females (Fig. 6C) is not
clear because there is no known direct mechanism of LH regulation by
inhibin. However, the inappropriate expression of inhibin may
contribute indirectly to the increase of LH. Exposing cultured thecal
cells to increased levels of inhibin has been shown to stimulate thecal
cell androgen production in vitro (39). The
local overexpression of inhibin in MT- transgenic mice may be
responsible for the increase in androgen production, as reflected by
increased T levels in transgenic females reported in the accompanying
paper. High levels of T may desensitize the pituitary and hypothalamus
to the negative feedback of the steroid hormones, causing increases in
LH. In women who suffer from virilizing congenital adrenal hyperplasia,
exposure to high levels of androgens at birth is associated with high
LH levels by the time they reach sexual maturity (40). If
MT- female mice are exposed to increased androgens during
development, this exposure could result in hypothalamic/pituitary
insensitivity and continued increases in serum LH.
In MT- transgenic male mice, the observed decrease in sperm numbers
may be secondary to increased inhibin or suppressed activin, and it is
likely to be a direct consequence of the altered gonadotropin
environment. The treatment of adult mice and hamsters with purified
inhibin has been shown to reduce spermatogonial numbers
(41). However, it is difficult to determine if inhibin
directly suppressed sperm production, because inhibin treatment also
slightly reduced serum FSH levels (41). Reduced or absent
FSH has been shown to decrease sperm numbers in rodents. For example,
FSH-deficient male mice exhibit a 75% reduction in sperm numbers but
are still fertile (42), and FSH receptor-deficient mice
similarly display a significant reduction in sperm numbers and remain
fertile (43). Thus, the likely basis for decreased sperm
production in MT- transgenic male mice is suppressed FSH levels.
Similarly, the observed reduction in MT- female ovulation rates is
probably attributable to the altered gonadotropin ratios in these
animals and not to a follicular defect (Fig. 8A). During the normal
estrus cycle, the GnRH-independent secondary FSH surge is important for
the recruitment of maturing follicles into the preovulatory pool
(23, 44). In MT- transgenic mice, total inhibin
-subunit levels are high and FSH levels are suppressed. The possible
reduction in FSH levels during the morning of estrus, the time of the
secondary FSH surge, could limit the number of immature follicles
recruited for subsequent ovulation. To determine if the ovulation rate
of MT- females could be rescued, we treated transgenic female mice
with exogenous gonadotropins. The exogenous gonadotropin stimulation of
MT- females resulted in an ovulation rate almost equal to that of
wild-type gonadotropin-stimulated females (Fig. 8B), suggesting that
reduced FSH, or the abnormal gonadotropin ratio, inhibits follicle
recruitment in MT- ovaries.
It is unlikely that reduced oocyte competence is responsible for
reduced MT- fertility. The combined decrease in birth rate of MT-
female mice compared with their wild-type littermates was 52%. The
combined reduction in the ovulation rate was 54%. Thus, the reduction
in offspring number mirrors the observed reduction in ovulation rates,
indicating that ovulatory defect is the primary factor in reduced
MT- female fertility.
The loss of the inhibin -subunit in inhibin-deficient mice results
in significant increases in serum activin levels (26),
indicating that the inhibin ß-subunits are up-regulated in the
absence of the -subunit. With the overexpression of the inhibin
-subunit in MT- transgenic mice, a decrease in inhibin
ß-subunit expression and/or activin dimer assembly might be expected.
We observed small reductions in ovarian ß-subunit protein expression
and in serum activin levels in older female mice. In contrast, in
situ analysis performed on ovaries from MT- transgenic mice
with an inhibin ßA riboprobe showed no
substantial change in ß-subunit RNA per follicle in the presence of
excess -subunit (Fig. 3). Activin-deficient mice (45, 46) exhibit a markedly different phenotype than MT-
transgenic mice. Although metallothionein is expressed early in
development (47), MT- transgenic mice mature normally,
whereas activin ßA-deficient mice or
ßA/ßB-deficient mice
die postnatally from cleft palate defects (46). Activin
ßB-deficient mice are viable, subfertile
because of a failure to nurse their young, and show a 20% increase in
serum FSH levels (45). This suggests that the phenotypes
of the MT- transgenic mice are more likely to be associated with the
increased inhibin levels than with decreased activin levels, although
both are likely to be contributory.
In conclusion, the overexpression of inhibin in transgenic mice
results in reduced serum FSH and an overall alteration in gonadotropin
ratios. MT- transgenic female mice exhibit reduced fertility as a
result of reduced ovulations, whereas MT- transgenic male mice are
fertile despite having significantly lower epididymal sperm numbers.
These findings support a role for inhibin and activin as key in
vivo modulators of FSH production, and they suggest that inhibin
and activin are essential for normal fertility and gametogenesis. The
accompanying paper focuses on potential autocrine/paracrine actions of
the inhibin transgene product within the ovary and describes the
effects of increased inhibin -subunit on gonadal morphology and
ovarian steroidogenesis in these transgenic mice.
|
Acknowledgments |
|---|
-subunit RIA reagents and antibodies and Dr. A.F. Parlow and the
National Hormone and Pituitary Program for FSH and LH RIA reagents.
Finally, we thank Dr. Jordan Shavit for his insight and assistance with
the ovulation analysis and Dr. Teresa Woodruff for her comments on the
manuscript and for recombinant human inhibin subunit proteins. |
Footnotes |
|---|
1 The first two authors contributed equally to this work. 
2 Present address: Division of Life Science, The Catholic University
of Korea, Puchon, South Korea 421-743.
3 Present address: Tularik Inc., South San Francisco, California
94080.
Abbreviations: hCG, Human CG; MT-, metallothionein-I
promoter inhibin -subunit; SSC, standard sodium citrate; TBS,
Tris-buffered saline.
Received July 26, 2000.
Accepted for publication July 18, 2001.
|
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, ßA,
and ßB subunits in various tissues predicts
diverse functions. Proc Natl Acad Sci USA 85:247–251-Inhibin gene expression occurs in the ovine adrenal cortex, and is
regulated by adrenocorticotropin. Mol Endocrinol 1:699–706, ßA and ßB subunits
in ovarian cell types during the rat estrous cycle. Mol Endocrinol 2:1352–1363-Inhibin is a tumour-suppressor gene with gonadal specificity
in mice. Nature 360:313–319[CrossRef][Medline]
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