Normal Prenatal but Arrested Postnatal Sexual Development of Luteinizing Hormone Receptor Knockout (LuRKO) Mice
Fu-Ping Zhang,
Matti Poutanen,
Johannes Wilbertz and
Ilpo Huhtaniemi
Department of Physiology (F.-P.Z., M.P., I.H.) Institute of
Biomedicine University of Turku FIN-20520, Turku, Finland
Department of Cell and Molecular Biology (J.W.) Karolinska
Institute S-17177 Stockholm, Sweden
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ABSTRACT
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To study further the role of gonadotropins
in reproductive functions, we generated mice with LH receptor (LHR)
knockout (LuRKO) by inactivating, through homologous recombination,
exon 11 on the LHR gene. LuRKO males and females were born
phenotypically normal, with testes, ovaries, and genital structures
indistinguishable from their wild-type (WT) littermates. Postnatally,
testicular growth and descent, and external genital and accessory sex
organ maturation, were blocked in LuRKO males, and their
spermatogenesis was arrested at the round spermatid stage. The number
and size of Leydig cells were dramatically reduced. LuRKO females also
displayed underdeveloped external genitalia and uteri postnatally, and
their age of vaginal opening was delayed by 57 days. The (-/-)
ovaries were smaller, and histological analysis revealed follicles up
to the early antral stage, but no preovulatory follicles or corpora
lutea. Reduced gonadal sex hormone production was found in each sex, as
was also reflected by the suppressed accessory sex organ weights and
elevated gonadotropin levels. Completion of meiosis of testicular germ
cells in the LuRKO males differs from other
hypogonadotropic/cryptorchid mouse models, suggesting a role for FSH in
this process. In females, FSH appears to stimulate developing follicles
from the preantral to early antral stage, and LH is the stimulus
beyond this stage. Hence, in each sex, the intrauterine sex
differentiation is independent of LH action, but it has a crucial role
postnatally for attaining sexual maturity. The LuRKO mouse is a close
phenocopy of recently characterized human patients with inactivating
LHR mutations, although the lack of pseudohermaphroditism in LuRKO
males suggests that the intrauterine sex differentiation in this
species is not dependent on LH action.
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INTRODUCTION
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The two gonadotropins, LH and FSH, have a key role in the
differentiation and maturation of mammalian sexual organs and
functions. After identification of genes for the gonadotropin subunits
and gonadotropin receptors (R), their mutations have been discovered in
males and females with various types of hypogonadism (for a review, see
Ref. 1). Mutations of the FSHß subunit gene cause infertility with
arrested follicular maturation in women and azoospermia in men.
Inactivating FSHR mutations in women cause the same phenotype as the
ligand mutations, but in men the phenotype is milder with only variable
impairment of spermatogenesis (1). Knockout models for both FSHß and
FSHR have been produced (2, 3, 4), and they display complete phenocopies
of the human FSHR mutations. A discrepant finding is the azoospermia
detected in the two men so far described with FSHß mutation (5, 6),
which is not found in the receptor or ligand knockout mice produced
(2, 3, 4) or in men with an inactivating FSHR mutation (7). Hence, the
necessity of FSH for spermatogenesis still remains controversial.
The consequences of inactivation of LH action also remains to be
clarified. Only a single man with LHß mutation has been reported (8);
he presented with normal sexual differentiation at birth but total lack
of postnatal sexual development. No women with such a mutation have yet
been described. Neither are there knockout models for LHß or LH
receptor (LHR). More is known about consequences of inactivating LHR
mutations in man (1). Depending on completeness of the receptor
inactivation, men present with pseudohermaphroditism ranging from mild
micropenis and hypo-spadias to complete sex reversal. The phenotype
in women is milder, including only anovulatory infertility.
In our exploration of the consequences of inactivation of
gonadotropin action, we concluded that a knockout mouse model for the
LHR would be a logical next step. It is known that LH stimulates Leydig
cell differentiation and steroidogenesis in the postnatal testis, but
its role in the fetal period is controversial (9). In the ovary, LH
stimulates theca cell androgen production, triggers ovulation, and
stimulates estrogen and progesterone production of corpus luteum. LH
actions in early stages of female development are unlikely, because
LHRs appear in the ovary only postnatally (10). In addition, there are
recent findings on LHR expression and LH actions in extragonadal organs
(11, 12). In addition to the expected phenotype of hypogonadism of the
LH receptor knockout (LuRKO) mice, the developmental and possible
extragonadal findings were especially hard to predict. Likewise,
it was interesting to see, to what extent FSH alone, in the absence of
LH action, was able to support gonadal function. We report here the
phenotypes of male and female mice with targeted disruption of the LHR
gene.
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RESULTS
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Gene Targeting
The purpose of the gene targeting was to eliminate exon 11
of the LHR gene (see Fig. 1
). After
electroporation of the targeting construct (Fig. 1A
) into ES cells, and
after drug selection, 250 surviving colonies were picked and screened
by PCR. Positive clones were further confirmed by Southern blots. Five
of the 250 clones gave a 2.3-kb fragment in PCR analysis, and bands
with expected size of 11 kb for the wild-type (WT) allele and 9 kb for
the targeted allele in Southern blot, indicating that homologous
recombination had occurred (Fig. 1
, B and C). Three of these cell lines
(C30, C76 and C101) were injected into blastocysts, yielding nine male
chimeras. The male chimeras were bred with C57BL/6 females, and
germline transmission (F1 offspring) was obtained with three of them.
Heterozygous mice were fertile and viable. Intercrossing the F1
heterozygotes yielded F2 progeny including 43 LHR WT (+/+) (22 female,
21 male), 86 heterozygous (+/-) (32 female, 53 male), and 37
homozygous (-/-) mice (16 female, 21 male), in agreement with the
expected Mendelian mode of inheritance.

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Figure 1. Targeted Disruption of the LHR Gene
A, The replacement targeting vector to delete exon 11 and part of
intron 10 of the LHR gene. The approximate locations of the PCR primers
used to screen for homologous recombinants and phenotype are shown
(arrows) with the original and predicted structure of the
gene after homologous recombination. B, Positive ES clones were
identified as homologous recombinations by PCR screen using the primers
(Neo1 and LHR3) shown in panel A. C, Representative of genomic DNA
isolated from two ES clones with homologous recombination and one WT ES
clone of cells, digested with HindIII, and analyzed by
Southern hybridization. The presence of 9- and 11-kb bands indicates
homologous recombination.
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Phenotype of the Animals
Homozygous LuRKO mice of both sexes were born phenotypically
normal. Testes of the newborn males were similar in size and
microscopic appearance (Fig. 2
) as those
of WT littermates, and their intraabdominal location adjacent to the
urinary bladder was the same as in WT males. Likewise, the internal
genitalia revealed under stereomicroscopy no difference between (+/+),
(+/-), and (-/-) males (data not shown). From about 3 weeks
postnatal age the male LuRKO mice were significantly lighter than (+/+)
or (+/-) mice of the same age, and at 45 days this weight difference
reached 30% (results not shown). No clear weight differences were
found between (-/-), (+/-), and (+/+) females up to 7 weeks of age
(data not shown). The LuRKO males could be distinguished from their
normal male littermates by external examination after 3035 days by
their small penis, short anogenital distance, and underdeveloped
scrotum. At the age of 7 weeks, their testes were very small [(+/+),
94 ± 6.9; (+/-), 91 ± 3.1; (-/-), 17 ± 0.77 mg;
mean ± SEM, n = 810] and located in the
abdominal cavity adjacent to the urinary bladder. The accessory
reproductive organs (seminal vesicles, epididymides, and prostates)
were macroscopically invisible (Fig. 2A
). Histology of the (-/-)
testes showed that seminiferous tubules were narrower and
spermatogenesis was arrested at the round spermatid stage (Fig. 2
). The
numbers and sizes of Leydig cells appeared dramatically decreased
as compared with (+/+) testes (Fig. 2
).

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Figure 2. Morphology and Histology of the Testes of Control
(+/+) and Homozygous (-/-) LuRKO Male Mice
A, Testes and accessory sex organs of a (-/-) and a (+/+)
littermate. VD, Vas deferens; SV, seminal vesicle; Epd, epididymis; BU,
bulbo-urethral gland. B and E, Testicular histology of a
1-day-old (-/-) and (+/+) mouse. C and F, Testicular histology of a
45-day-old (-/-) and (+/+) mouse. D and G, As in panels C and F, at
higher magnification. Arrows indicate round spermatids. The
bar in panels BG is 100 µm.
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In female (-/-) mice, the age of vaginal opening was delayed to
3538 days, compared with 3032 days in WT mice. Internally, the
ovaries were about 50% reduced in size, and the uteri were
significantly thinner (Fig. 3A
). Ovarian
histology at the age of 7 and 12 weeks showed in (-/-) mice presence
of follicles up to the early antral stage, but no preovulatory
follicles or corpora lutea (Fig. 3
). No apparent differences were seen
in the thickness of the theca cell layers surrounding the developing
follicles. The uterine histology showed thinning of all cell layers and
absence of glandular structures (Fig. 3
). Comparison of estrous cycles
of the (+/+) and (-/-) mice at the age of 12 weeks showed the cyclic
periodicity with recognizable estrus every 4 days in (+/+) mice, but
not in (-/-) mice.

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Figure 3. Morphology and Histology of Ovaries and Genital
Organs of Control (+/+) and Homozygous (-/-) LuRKO Female Mice
A, Ovaries, uteri, and vagina of a (-/-) and a (+/+) littermate.
B and E, Ovarian histology of a 7-week-old (-/-) and (+/+)
mouse. C and F, As in panels B and E, at higher magnification. D
and G, Uterine histology of a (-/-) and (+/+) mouse. CL, Corpus
luteum. The bar in panels BG is 100 µm.
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No differences were found between (+/-) and (+/+) mice in any of the
parameters studied.
Analysis of the Disrupted LHR Gene
The targeting construct was designed so that the pGKneo
insert would replace exon 11, which encodes the transmembrane and
cytoplasmic receptor domains, and part of the 3'-extracellular domain,
thus preventing the formation of full-length functional LHR capable of
anchoring to the plasma membrane and of signal transduction. However,
it is possible that the remaining fragment of the LHR gene could
be transcribed into truncated forms of LHR mRNA. We performed RT-PCR
analysis with different primer pairs on testicular and ovarian RNA from
LuRKO and WT mice. Using primer pair LHRm1 and 2 (specific for the
extracellular domain of LHR) an amplicon of 412 bp was detected from
(+/+), (+/-), and (-/-) testes and ovaries (Fig. 4
). Using primer pair LHRm3 and 4
(specific for transmembrane and cytoplasmic domains), a 359-bp band was
found in (+/+) and (+/-) mouse testes and ovaries, but not in those of
(-/-) mice (Fig. 4
). Northern hybridization analysis of testicular
RNA by using a cRNA probe specific for extracellular domain of LHR
revealed four major transcripts of LHR mRNA with sizes of 6.9, 2.6,
1.7, and 1.2 kb in the (+/+) and (+/-) mice, but only one band of 1.2
kb in the (-/-) mice. When using the cRNA probe specific for the
transmembrane domain of LHR, two major bands with sizes of 6.9 and 2.6
kb were present in the (+/+) and (+/-) mice, but no hybridization was
observed in the (-/-) samples (data not shown). Hence, the LuRKO
mice do not synthesize any mRNA encoding the full-length functional
LHR.

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Figure 4. RT-PCR Detection of LHR mRNA in WT (+/+),
Heterozygous (+/-), and LHR Deficient (-/-) Mice Testis and Ovary
A, PCR products generated by using a primer pair specific for the
extracellular receptor domain. B, PCR products generated by using a
primer pair specific for the transmembrane, cytoplasmic, and 3'-part of
extracellular domains. C, PCR products using primers specific for
ß-actin mRNA.
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hCG Binding
To confirm whether the LHR-deficient mice express functional
or truncated LHR, radioligand receptor assay was carried out using both
testicular membranes and detergent-solubilized testicular homogenates.
The results indicated that (+/+) and (+/-) mice expressed high levels
of specific [125I]iodo-hCG binding in both
testis membranes and detergent-solubilized extracts, whereas none was
detected in either samples from the (-/-) mouse testes (Fig. 5
).

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Figure 5. Specific [125I]iodo-hCG Binding in
Control (+/+), Heterozygous (+/-), and LHR-Deficient (-/-) Adult
Mice (7 weeks old) to Testis Membranes (left panel) and
Detergent Extracts (right panel)
The mean binding measured in (+/+) testes was assigned a value of
100%. Each bar is the mean ± SEM
of three samples. The binding in (-/-) samples was indistinguishable
from zero, and it differed from (+/+) samples at P
< 0.001.
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Analysis of Cholesterol Side-Chain Cleavage P450
(P450scc) and Cytochrome P-450 17-Hydroxylase
(P450 17-OH) Gene Expressions
To further characterize the potential residual activities of
steroidogenesis of Leydig and theca cells of the LuRKO mice, we
analyzed the expression of the P450scc and P450 17-OH mRNAs. P450scc,
converting cholesterol to pregnenolone, is a key rate-limiting step in
Leydig cell testosterone biosynthesis. Northern blot analysis indicated
that P450scc mRNA was dramatically decreased (>20 fold) in LuRKO,
compared with WT testes (Fig. 6
). P450
17-OH is a theca cell-specific enzyme and necessary for their androgen
production. In the LHR-deficient ovaries, the P450 17-OH mRNA was
decreased by 12-fold as compared with the expression of WT ovaries
(Fig. 6
). These data provide evidence that the LHR gene deletion
profoundly, although not totally, reduces the steroidogenic activities
of the Leydig and theca cells.

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Figure 6. Analysis of Gene Expressions in Control (+/+) and
LHR-Deficient (-/-) Mice
A, Northern blot analysis of P450scc gene expression in
testis samples. B, Northern blot analysis of P450 17-OH gene expression
in ovarian samples. The lower panels are the 28S
ribosomal RNA loading controls.
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Gonadotropin and Steroid Assays
Gonadotropin assays (Fig. 7
)
indicated that serum LH levels were dramatically increased in (-/-)
males and females, whereas there were no significant difference of
pituitary LH levels between the (+/+), (+/-), and (-/-) mice. Serum
FSH levels were elevated, and pituitary FSH levels were decreased in
(-/-) males and females as compared with (+/-) and (+/+) males and
females. Serum and testicular testosterone levels of (-/-) males were
significantly decreased (Fig. 8
) as
compared with (+/+) and (+/-) males. However, the former levels were
somewhat higher than we detect in castrated mouse circulation (13),
indicating that testicular steroidogenesis was not totally abolished by
LHR inactivation. Likewise, the ovarian estradiol concentration was
significantly decreased in (-/-) female mice compared with (+/+) and
(+/-) females (Fig. 8
). Similar suppression of ovarian progesterone
was observed in the LuRKO females (data not shown).

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Figure 7. Gonadotropin Levels in WT (+/+), Heterozygous
(+/-), and LHR-Deficient (-/-) Adult Mice
A, Serum and pituitary LH levels of 7-week-old male and female
mice. B, Serum and pituitary FSH levels from 7-week-old male and
female mice. *, P < 0.05; and **, P <
0.01, compared with (+/+) and (+/-) groups. n = 610 mice per
group.
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Figure 8. Testosterone and Estradiol Levels in WT (+/+),
Heterozygous (+/-), and LHR-Deficient (-/-) Adult Mice
Testicular (A) and serum testosterone (B) levels of 7-week-old male
mice. C, Ovarian estradiol level of 7-week-old female mice. **,
P < 0.01; and ***, P < 0.001,
compared with (+/+) and (+/-) groups. n = 610 mice per group.
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The small seminal vesicles and prostates of the (-/-) males, and thin
uteri of the (-/-) females, provide further functional evidence for
long-term suppressed sex steroid production in LuRKO mice.
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DISCUSSION
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Targeted disruption of exon 11 of the LHR gene resulted in
complete loss of LHR binding in both intact cells and
detergent-solubilized extracts from the LHR-deficient mouse testes.
RT-PCR analyses demonstrated the absence of exon 11-specific mRNA
sequences in testes and ovaries, whereas those encoding the
extracellular domain of the receptor were transcribed. Since the
extracellular part of the receptor is unable to anchor to the plasma
membrane and does not have the structures needed for signal
transduction (14), it cannot exert regulatory functions alone even if
secreted into the extracellular space. Admittedly, modulatory effects
of truncated LHR forms on function of the full-length receptor have
been proposed (15), but in the absence of the latter these are not
possible in the LHR-deficient mice. Furthermore, our data showed that
no ligand binding was found in both intact cells and
detergent-solubilized extracts from the LHR-deficient mouse testes.
Previous studies have demonstrated that
[125I]iodo-hCG binding could detect truncated
LHR species in detergent-solubilized extracts (14). For this reason,
the knockout model produced represents total inactivation of LHR
function.
Interestingly, the sexual differentiation and gonadal histology of
female and male LuRKO mice were indistinguishable from WT littermates
at birth. In females, this is not surprising, since the early female
sex differentiation is known to be independent of ovarian function
(16), and gonadotropin receptors occur in rodent ovaries only several
days after birth (17). Although fetal testicular testosterone synthesis
is crucial for male sexual differentiation, the LuRKO mice provide, for
the first time, direct evidence that specific elimination of LH action
does not hamper this function. This has been suggested indirectly by
earlier findings on normal intrauterine masculinization of
gonadotropin-deficient hpg (18) and common
-subunit
knockout (19) mice, and on unmeasurable levels of LH in rat fetal
circulation at the time of the sharpest increase in fetal testicular
testosterone production (20).
The testes of the LuRKO mice weighed about 17 mg, which were
about 18% of that of WT testes, but 5-fold more than those of the
hpg mice (18, 21). This increased weight over hpg
mice can be ascribed to the elevated FSH action in the LuRKO mice,
because this gonadotropin is known to stimulate Sertoli cell
proliferation in the neonatal testis (22, 23, 24). While the
spermatogenesis of hpg and common
-subunit knockout mice
proceeds up to the diplotene stage (18, 19), some tubules of the LuRKO
mice show round spermatids, i.e. completion of meiosis. This
is supported by the findings that progression of spermatogenesis from
spermatocytes to spermatids can be stimulated after hypophysectomy by
either FSH or androgen (25). However, both the intraabdominal location
and insufficient testosterone production of the LuRKO testes offset
further progression of spermatogenesis. Interestingly, it is typical
for experimental cryptorchidism with undisturbed Leydig cell
testosterone production (26), including the recently developed Insl3
knockout mouse (27), that they, despite normal androgen levels, lack
postmeiotic germ cells. This raises the possibility that normal
intratesticular testosterone concentration in the abdominal temperature
is deleterious to spermatogenesis, as seems to occur during the
recovery of spermatogenesis after cytotoxic or radiation insults
(28).
Concerning testicular descent, no difference was found at birth
in the location of the testes, adjacent to the urinary bladder, between
the (-/-) and (+/+) mice. Hence, the lack of LH stimulation in
utero did not hamper the first transabdominal phase of testicular
descent, known to be dependent on both androgen and Insl3 (29).
The ovaries of the LuRKO mice were reduced in size and histological
analysis revealed follicles up to the early antral stage, but no
preovulatory follicles or corpora lutea. The ovaries of hpg
mice have follicles up to the preantral stage (18), which indicates
that FSH action, present in LuRKO mice, has a distinct effect on
progression of preantral follicles to the early antral stage.
Correspondingly, the lack of preovulatory follicles and corpora lutea
indicates that the very last steps of follicular maturation, as well as
ovulation, do not occur without LH action. Another intriguing feature
of the LuRKO ovaries was the apparent normal thickness of thecal cell
layers surrounding the follicles. Hence, although theca cells are a
target of LH action, their survival is apparently not dependent on LH,
which observation can also be made in hpg ovaries (18).
However, theca cell androgen production, to provide substrate for
granulosa cell estrogen production, is LH dependent. Therefore, the
defective estrogenization of LuRKO females was expected, as
demonstrated by their low ovarian estradiol level, delayed sexual
maturation, and hypoplastic uteri.
As an indicator of Leydig cell steroidogenic activity (30, 31),
we measured the mRNA level of P450scc in the
LuRKO testes. The low but detectable level of expression indicates that
low constitutive expression of this enzyme is possible in the absence
of LH action in the precursor Leydig cells detected in the LuRKO
testes. In accordance, the serum testosterone levels in the LuRKO males
were slightly higher than measured by us in orchidectomized mice (13).
However, the physiological significance of this residual androgen
production is unlikely in view of the lack of postnatal sexual
development of the LuRKO males. Likewise, low but detectable levels of
P450 17-OH mRNA, a marker of theca cell steroidogenesis (32), was
detected in the LuRKO ovaries, indicating that this enzyme is also
expressed constitutively at low levels. This finding, together with the
well developed theca cells and low but detectable estradiol level of
the LuRKO ovaries, explains the delayed vaginal opening of the LuRKO
females.
Both female and male LuRKO mice represent close phenocopies of the
respective human mutation (1). LHR inactivation in males causes
pseudohermaphroditism of varying severity. The most severe forms
present with female genitals, absence of uterus, low testosterone, and
high LH. The milder forms, with partial LHR inactivation, display a
broader array of phenotypes ranging from micropenis to hypospadias. The
severity of the phenotype has been shown to correlate with the degree
of LHR inactivation (1). Although the receptor inactivation in LuRKO
mice is total, the male phenotype is less dramatic than in connection
with similar human mutations. This indicates that the gonadotropin-
independent component of fetal Leydig cell androgen production is more
prominent in the mouse than in the human. The presence of chorionic
gonadotropin (hCG) in human fetal circulation may explain the
difference. Regulation of the critical process of testosterone-
dependent male sexual differentiation needs, in addition to pituitary
LH, a backup mechanism, which is hCG in the human (33) and testicular
paracrine regulation (20) in the rodent.
The female phenotype of LuRKO mice is even closer to that of the
inactivating human LHR mutations (1). Affected women have normal
primary and secondary sex characteristics, increased gonadotropins, and
low estrogen and progesterone production. Likewise, suppressed but not
absent estrogen production of the LuRKO mice is reflected by the
presence of granulosa cells in their ovaries, delayed vaginal opening,
and hypoplastic uteri. Ovarian histology demonstrates follicles at
early stages of development, but no preovulatory follicles or corpora
lutea, in both women with LHR inactivation and in LuRKO mice. These
observations in the human and mouse support the view that LH is
essential for normal estrogen production and ovulation, whereas
follicular development is initially independent of gonadotropins and,
in its final stages, is dependent on FSH and LH.
In conclusion, the LuRKO mouse allows us to identify directly the
specific LH- dependent steps of male and female sexual differentiation
and adult gonadal functions. It is a close phenocopy of completely
inactivating mutations of the human LHR gene, and it provides a
valuable tool for experimental studies of pathogenesis of this
condition. Although all effects characterized in the present study were
concerned with development and function of gonads and sex organs, the
LuRKO model also helps us to explore the putative and recently
documented extragonadal actions of LH (11, 12), which most notably
apply to the tumorigenic effects observed with this hormone (34, 35).
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MATERIALS AND METHODS
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Genomic Clone Isolation
A 9.8-kb genomic BamHI/BamHI fragment on
the mouse LHR gene, containing exon 11, 4.7 kb of the flanking intron
10 sequences and 4.0 kb of flanking 3'-sequences was isolated from a
mouse ES129/SvJ BAC library (Genome Systems, St. Louis,
MO), using a cDNA probe specific for exon 11 of the rat LHR gene (36)
(Fig. 1
). Sequencing confirmed validity of the genomic fragment.
Targeting Vector Construction
The targeting plasmids pKO Scramber913, pKO SelectNeo V800, and
pKO SelectTK V830 were purchased from Lexicon Genetics, Inc. (The
Woodlands, TX). Briefly, the targeting vector contained a 4.2-kb
BamHI-XbaI fragment as the 5'-homology region and
a 2.2-kb EcoRI-BamHI fragment representing the
3'- homology region, a positive selection marker (the PGK-Neo
expression cassette), and an MC1-tk (thymidine kinase) expression
cassette (Fig. 1
).
Embryonic Stem (ES) Cell Culture
The ES cell line, AB.2.2-prime ES cells, was purchased
from Lexicon Genetics, Inc. (The Woodlands, TX) and cultured on
neomycin-resistant primary embryonic fibroblast feeder layers
irradiated with 3000 rads. Ten million cells were electroporated (500
µF, 240 V) with 30 µg of linearized targeting construct. After
electroporation, the surviving cells were plated on 100-mm diameter
culture dishes and exposed to G418 (Sigma) at 300 mg/liter
and 1 µM of ganciclovir (Hoffman-LaRoche Inc., Basle, Switzerland) for 910 days. Colonies were picked
into 24-well plates and grown for 56 days, when about one eighth of a
colony was replated onto 24-well plates for genomic DNA extraction,
with the remainder being frozen at -80 C.
Screening of Targeting Clones
DNA was isolated from each individual clone and screened by PCR,
which produced a 2.3-kb amplicon with a primer pair corresponding to
the 3'-end of pGKneo (Neo1, 5'-GGGCTCTATGGCTTCTGAGGCGGA-3') and to the
flanking 3' end of exon 11 (LHR3, 5'-TCTCAGGGAGGATTTGGGTATGG-3') (Fig. 1A
). Correct targeting of the ES cells was further confirmed by
Southern hybridization analysis of HindIII- digested ES
cell genomic DNA and a probe specific for the flanking sequence of
intron 10; the expected band from unmodified LHR was 11 kb in size and
9 kb for the deleted LHR gene.
Mouse Breeding
The targeted ES cells were injected into blastocysts from
C57BL/6J females and implanted into pseudopregnant mothers to proceed
to term. Chimeras were identified by coat color, and males were bred to
C57BL/6J females to test germline transmission. Genotyping of the mice
was carried out by using PCR on genomic DNA with primer pairs for the
WT allele (LHR1, 5'-TCTGGGGATCTTGGAAATGA-3'; LHR2,
5'-CACCTTGACACCTGGAGT-3') and for the targeted allele (Neo1-LHR2) (Fig. 1
). Tail DNA from F1 offspring with agouti coat color was screened by
PCR with primer pair Neo1 and LHR2. F2 offspring and the subsequent
generations were screened by PCR using primer pairs for detecting
presence of the targeted LHR gene and the WT gene.
All mice were handled in accordance with the institutional animal care
policy of the University of Turku.
Histological Analysis
Testes, ovaries, epididymides, seminal vesicles, uteri, and
pituitary glands were removed, fixed in 4% paraformaldehyde at 4 C for
414 h, dehydrated, and embedded in paraffin, and sectioned at 5 µm
thickness. Sections were stained with Harris hematoxylin and eosin (BDH
Ltd., Poole, UK). The reproducibility of all the morphological data was
verified by similar findings in at least three different animals.
RNA Isolation and Analyses
RNA isolation and RT-PCR were carried out as previously reported
(37). RNA was extracted by the single-step method (38). cDNA was
generated by reverse transcriptase (RT) from 2 µg of testicular or
ovarian RNA, using avian myeloblastosis virus (AMV)-RT with
random hexamers (Promega Corp., Madison, WI), in a final
volume of 25 µl. Subsequent PCR analysis was performed on 3 µl of
the cDNA and 0.1 µl of [32P]-CTP (
400
Ci/mmol; Amersham Pharmacia Biotech, Aylesbury, UK ), and
the PCR products were analyzed by electrophoresis on 1.4% agarose
gels. The oligonucleotide primers used for RT-PCR were designed
according to the published cDNA sequences of mouse LHR (39). PCR
amplification with the primer pair LHRm1 and 2 (LHRm1,
5'-TGAACCCGGTGCTTTTACAA-3'; LHRm2, 5'-CGTGGCGATGAGCGTCTGAATG-3'),
specific for the extracellular domain of LHR, yields a 412-bp fragment.
With the primer pair LHRm3 and 4 (LHRm3,
5'-ATCGCCACGTCATCCTACTCACTG-3'; LHRm4, 5'AGCCAAATCAACACCCTAAG-3'),
specific for exon 11 of LHR, a 359- bp amplicon is produced.
Northern Hybridization Analysis
Ten or five micrograms of total RNA from testis and ovary were
resolved on 1.2% formaldehyde denaturing agarose gel and transferred
onto nylon membrane (Hybond-XL, Amersham Pharmacia Biotech). Prehybridization and hybridization were performed as
previously described (37). Briefly, the filters were prehybridized for
at least 4 h at 65 C in a solution containing 50% formamide,
3 x SCC (1 x SCC = 150 mM NaCl and 15
mM sodium citrate, pH 7.0), 5 x Denhardt solution,
1% SDS. Hybridization was carried out at 66 C overnight in the same
solution after adding the [32P]-labeled cRNA
probe. After hybridization, the membranes were washed twice with 1
x SCC and 0.1% SDS at 65C for 30 min each time and twice with 0.1 SCC
and 0.1% SDS at 66 C for 30 min each time. The membranes were exposed
to x-ray film (Kodak XAR-5, Eastman Kodak Co., Rochester,
NY) at -70 C for 13 days. The molecular sizes of the mRNA species
were estimated by comparison with mobility of the 18S and 28S ribosomal
RNAs. The [32P]-labeled cRNAs were synthesized
using a Riboprobe synthesis II kit (Promega Corp.),
[32P]UTP (Amersham Pharmacia Biotech), and the corresponding cDNA templates. For generation
of P450scc riboprobe, a template composed of a
fragment of rat P450scc cDNA (spanning bp
186695), subcloned into T vector under T7 RNA polymerase promoter,
was used (40). Antisense cRNA probe for P450 17-OH mRNA analysis was
produced using as template a fragment of mouse P450 17-OH cDNA
(spanning bp 55616) subcloned into T vector under the T7 RNA
polymerase promoter (41). For the LHR cRNA probes, the cDNAs used as
templates corresponded to bases 441849 of extracellular domain of rat
LHR cDNA (36), and to bases of 1,0021,461 of transmembrane domain of
mouse LHR cDNA (36), respectively.
hCG Binding Assay
Testicular LHR binding was measured as previously reported (42, 43). Briefly, a piece of testis tissue was homogenized with an
Ultra-Turrax 18/10 homogenizer in Dulbeccos PBS + 0.1% BSA (0.5 mg
tissue/ml). Highly purified hCG (NIH CR-125; 13,000 IU/mg) was
radioiodinated using a solid phase lactoperoxidase method.
One-hundred-microliter aliquots of testicular homogenate were incubated
in triplicate at room temperature for 18 h, in the presence of a
saturating concentration (150,000 cpm;
3 ng) of
[125I]iodo-hCG. Nonspecific binding was
assessed in the presence of a 1,000-fold excess of unlabeled hCG
(Pregnyl, Organon, Oss, The Netherlands). The
centrifugation step used to separate bound and free hormone (1000
x g, 30 min at 4 C) precipitates only membrane-bound
receptors (42).
To measure hCG binding to detergent-solubilized (i.e.
membrane-bound and soluble) receptors, the testes were homogenized in
ice-cold buffer A (150 mM NaCl, 20
mM HEPES, pH 7.4) containing 20% glycerol, 1%
Nonidet P-40 (NP-40), and protease inhibitor cocktail
(Sigma), and incubated on ice for 30 min. After
centrifugation at 13,000 rpm for 30 min at 4 C, the supernatant was
used for the ligand binding assay. The binding reaction was carried out
as above, except that the incubation was overnight at 4 C. Free and
bound [125I]iodo-hCG were separated by
precipitating the samples with polyethylene glycol (mol wt 8,000). Each
tube received 0.2 ml of 5 g/liter solution of bovine
-globulin in
buffer A and 0.5 ml of 30% (wt/vol) polyethylene glycol in buffer A.
After incubation at 4 C for 10 min, the samples were pelleted at 2,800
rpm for 30 min, and supernatants were removed. Pellets were resuspended
in 0.9 ml of buffer A containing 0.1% NP-40 and 20% glycerol. After
addition of 0.5 ml of polyethylene glycol, the tubes were mixed,
incubated at 4C for 10 min, and centrifuged again. The supernatants
were aspirated, and the pellets were counted in a
-spectrometer.
Nonspecific binding was determined in these measurements as above.
Hormone Measurements
Serum and pituitary LH and FSH levels were determined by
immunofluorometric assays as earlier described (44, 45).
Intratesticular testosterone and ovarian estradiol and progesterone
were determined by homogenizing one (-/-) testis and a weighed
portion (approximately half) of one (+/+) or (+/-) testis in 0.5 ml
PBS, and a pair of ovaries in 0.2 ml PBS. One hundred microliters of
the gonadal homogenates or serum were extracted twice in 2 ml diethyl
ether and evaporated to dryness overnight in a fume hood. After
reconstitution into PBS, testosterone and progesterone were measured by
standard RIAs. Estradiol level was measured by a DELFIA Estradiol kit
(Wallac, Inc., Turku, Finland) according to the
manufacturers instruction. Protein concentrations in homogenates were
measured using the Bradford method (46).
Statistical Analysis
The Statview program (Windows version 4.57; Abacus Concepts
Inc., Berkeley, CA) was used for ANOVA and t tests.
Significance was set as P < 0.05. The values are
presented as mean ± SE.
 |
ACKNOWLEDGMENTS
|
---|
We thank F. Zhu, T. Laiho, J. Vesa, and N. Messner for their
skillful technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Professor Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi.
This study was supported by grants from the Academy of Finland and The
Sigrid Jusélius Foundation.
Received for publication June 7, 2000.
Revision received September 25, 2000.
Accepted for publication October 2, 2000.
 |
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