Targeted Disruption of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene
Z. M. Lei,
S. Mishra,
W. Zou,
B. Xu,
M. Foltz,
X. Li and
Ch. V. Rao
Division of Basic Science Research Department of Obstetrics and
Gynecology University of Louisville Health Sciences Center
Louisville, Kentucky 40292
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ABSTRACT
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LH/hCG receptors were disrupted by gene
targeting in embryonic stem cells. The disruption resulted in
infertility in both sexes. The gonads contained no receptor mRNA or
receptor protein. Serum LH levels were greatly elevated, and FSH levels
were moderately elevated in both sexes; estradiol and progesterone
levels decreased but were not totally suppressed in females;
testosterone levels were dramatically decreased and estradiol levels
moderately elevated in males. The external and internal genitalia were
grossly underdeveloped in both sexes. Abnormalities included
ambiguous vaginal opening, abdominal testes, micropenis,
dramatically decreased weights of the gonads and reproductive tract,
arrested follicular growth beyond antral stage, disarray of
seminiferous tubules, diminished number and hypotrophy of Leydig
cells, and spermatogenic arrest beyond the round spermatid stage.
LH/hCG receptor gene disruption had no effect on FSH receptor mRNA
levels in ovaries and testes, progesterone receptor (PR) levels in
ovaries and androgen receptor (AR) levels in testes. However, it caused
a dramatic decrease in StAR and estrogen receptor-
(ER
) mRNA
levels and an increase in ERß mRNA levels in both ovaries and testes.
Estradiol and progesterone replacement therapy in females and
testosterone replacement in males, to determine whether
phenotype and biochemical changes were a consequence of decreased
gonadal steroid levels or due to a loss of LH signaling, revealed
complete restoration of some and partial restoration of others.
Nevertheless, the animals remained infertile. It is anticipated that
the LH receptor knockout animals will increase our current
understanding of gonadal and nongonadal actions of LH and hCG.
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INTRODUCTION
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LH from anterior pituitary gland and human (h)CG from human
placenta are glycoprotein hormones that belong to a family that
includes FSH and TSH (1). LH and hCG also belong to cystine-knot growth
factors family, which includes nerve growth factor, platelet derived
growth factor-ß, and transforming growth factor-ß (2). Members of
glycoprotein hormone family are heterodimers of noncovalently bound
- and ßsubunits (1). The
-subunit is identical, whereas
ß-subunits, which specify hormone specificity, are different among
these hormones except LH and hCG (1). These two hormones have similar
but not identical ß-subunits (1). Structural homology between LH and
hCG makes them functionally similar (1). The functional similarity
comes from the fact that both hormones bind to the same receptors (3, 4). These receptors are single-chain transmembrane glycoproteins that
belong to the G protein-coupled receptor family (3, 4). Members of this
family have an extracellular hormone binding domain, seven
transmembrane spanning regions, and an intracellular region that couple
to G proteins (3, 4). The LH/hCG receptor is encoded by a single-copy
TATA-less gene (5). It spans more than 70 kbp containing 11 exons and
10 introns (5, 6, 7). The first 10 exons encode the extracellular hormone
binding domain, and the last exon encodes the rest of the receptor
(5, 6, 7). The transcription is initiated from multiple sites present 50
to 450 nucleotides upstream from the translation start site (5, 6, 7). As
a result and also due to differences in polyadenylation and alternate
splicing, virtually every LH/hCG receptor-positive tissue/cell contains
multiple transcripts (8, 9, 10).
In addition to gonads, which contain a high abundance of receptors (1, 11, 12, 13, 14), a number of nongonadal tissues (i.e. female and
male reproductive tract, fetoplacental unit, brain, adrenal zona
reticularis, skin, breast, urinary bladder, etc.) also contain low
levels of functional LH/hCG receptors (10, 15, 16, 17, 18, 19, 20, 21, 22). The gonadal actions
of LH and hCG result in an increased synthesis of steroid hormones in
the body, which act on multiple targets, including gonads
themselves (1, 11, 13, 14, 23, 24, 25, 26). Nongonadal actions of these
hormones are diverse and vary with the organ and its physiological
state (10, 15, 16, 17, 18, 19, 20, 21, 22). There is an unavoidable degree of uncertainty
concerning the role of LH vs. the role of other hormones in
different functions of gonadal and nongonadal tissues. This is due to
different hormones acting both sequentially and synergistically. Our
long-term goal is to advance current understanding of the total actions
of LH and hCG in the body. Toward this goal, we have developed mice in
which LH/hCG receptors were completely inactivated by gene targeting in
embryonic stem cells. The phenotypes of these animals were
characterized, and the effect of steroid hormone replacement therapy on
the phenotype reversal was tested.
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RESULTS
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Generation of LH/hCG Receptor Knockout Mice
A single gene with multiple transcription initiation sites present
in the 5'-flanking region encodes multiple transcripts and usually a
single protein in virtually every LH/hCG receptor-positive tissue in
the body (8, 9, 10). To ensure that the gene is completely inactivated,
targeting vector was constructed so that a part of the 5'-flanking
region containing the promoter region and multiple transcription
initiation sites, as well as most of exon 1, would be deleted upon
homologous DNA recombination in the host cell. Predicted from the
restriction sites in the targeting construct, recombination events in
two alleles should give only a 10 kbp fragment by Southern blotting
with probe A on genomic DNA digested with StuI. Animals
containing one wild-type allele (+/-) should also give an additional
8-kbp fragment. Figure 1B
shows that this
expectation was met. Further analysis with B or neomycin probes on
genomic DNA digested with SphI also confirmed the disruption
of LH/hCG receptor gene and integration of neomycin gene into the host
cell genome with no rearrangements or extra insertional events (data
not shown).

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Figure 1. Generation of LH/hCG Receptor Gene Knockout Mice
A, Construction of a targeting vector. Four kilobase pairs of the
LH/hCG receptor gene (St through X) that contained the promoter region
and most of exon 1 sequence (dark bar) were replaced
with the neomycin resistance gene cassette. Two
arrowheads indicate opposite orientation of neomycin and
thymidine kinase genes from transcription of the LH/hCG receptor gene.
Two DNA probes designated as A and B were used for screening clones of
embryonic stem cells with homologous DNA recombination. Restriction
enzyme sites: E, EcoRI; Ev, EcoRV; Sp,
SphI; St, StuI, X, XhoI.
B, Southern blot analyses of DNA isolated from tail biopsies. DNA was
digested with StuI and hybridized with A probe. An 8-kbp
wild-type LH/hCG receptor allele and a 10-kbp mutant allele in both
+/- and -/- mice are shown. C, Nonquantitative RT-PCR for LH/hCG
receptor mRNA. A predicted 437-bp LH/hCG receptor fragment was
amplified from total RNA of +/+ and +/- mice gonads. This fragment was
not detected in -/- mice gonads despite amplification of GAPDH mRNA.
D, Specific 125I-hCG binding to ovaries and testes. *,
P < 0.05; and **, P < 0.0001,
compared with corresponding wild-type littermates. The data presented
were the means ± SEs of measurements on three
animals. E, Semiquantitative RT-PCR for FSHR, ER , ERß, PR, StAR,
and AR mRNAs in mouse gonads. The relative abundance of mRNA levels in
+/+, +/-, and -/- mice gonads was determined by scanning the density
of each band and expressing as ratios with ß-actin. The ratio for
each mRNA species in +/+ mice was set at 1 for calculating changes in
+/- and -/- littermates. The blots presented are representative and
the fold changes (means ± SEs) were calculated from
three independent experiments.
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Figure 1C
shows that while LH/hCG receptor mRNA could be detected in
gonads of +/+ and +/- animals by RT-PCR, it was undetectable in gonads
of -/- littermates despite the amplification of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of
expression changes in genes that are important for gonadal function by
semiquantitative RT-PCR demonstrated that targeted disruption of the
LH/hCG receptor gene had no effect on FSH receptor mRNA levels in
ovaries and testes, progesterone receptor (PR) mRNA levels in ovaries,
and androgen receptor (AR) mRNA levels in testes (Fig. 1D
). However, it
caused a dramatic decrease in steroidogenic acute regulatory protein
(StAR) and estrogen receptor-
(ER
) mRNA levels and an increase in
ERß mRNA levels in both ovaries and testes.
Immunostaining for LH/hCG receptor protein revealed high levels in
thecal cells, followed by granulosa and luteal cells in the ovaries of
+/+ and +/- animals (Fig. 2
, a and b).
In testes, Leydig cells contain the highest receptor immunostaining
(Fig. 2
, e and f). In addition, different stages of spermatogenic cells
also contained some receptor immunostaining, which is in agreement with
earlier studies demonstrating that epididymal and ejaculated sperm
contain functional LH/hCG receptors (22, 27, 28). Receptor
immunostaining was absent in procedural controls performed on gonads
from +/+ animals (Fig. 2
, d and h). In contrast to +/+ and +/-
animals, gonads of -/- littermates had no detectable receptor
immunostaining (Fig. 2
, c and g). The lack of receptor protein in the
gonads of -/- animals is further confirmed by ligand binding studies
demonstrating the absence of 125I-hCG binding in
contrast to +/+ and +/- littermates (Fig. 1E
). While ovaries contained
the same level of binding, testes showed a modest decrease in +/-
animals compared with +/+ littermates.

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Figure 2. Immunocytochemistry for LH/hCG Receptors in Ovaries
(a, b, c, and d) and Testes (e, f, g, and h) from +/+ (a, d, e, and h),
+/- (b and f), and -/- (c and g) Mice
Arrows show strong receptor immunostaining in theca and
Leydig cells. Moderate to low receptor immunostaining is also seen in
granulosa and luteal cells, spermatogonia, and spermatocytes. Panels d
and h are immunostaining controls in which receptor antibody was
preabsorbed with excess receptor peptide. FL, Follicle; CL, corpus
luteum; ST, seminiferous tubule. Magnification, 300x.
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Female and Male Phenotype in LH/hCG Receptor Knockout Animals
The vaginal opening was quite ambiguous in -/- animals (Fig. 3A
). The ovaries were small and pale and
the reproductive tract was very thin compared with +/+ and +/-
littermates at 60 days of age (Fig. 3B
). The wet weights of the
reproductive tract and ovaries dramatically decreased in -/- animals
compared with +/+ and +/- littermates (Fig. 3C
).

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Figure 3. Female and Male Phenotype in LH/hCG Receptor Gene
Knockout Mice
A, Appearance of external female genitalia. Vaginal orifice was
difficult to detect in -/- compared with +/+ and +/- littermates. B,
Gross appearance of ovaries and reproductive tract. Only one ovary is
shown; the other was removed for analysis. Ovaries were small and pale,
and the reproductive tract was thin in -/- mice compared with +/+ and
+/- littermates. C, Wet weight of reproductive tract and ovaries. *,
P < 0.001 compared with corresponding tissues from
+/+ and +/- littermates. The data presented were the means ±
SEs of measurements on three animals. D, Appearance of
external male genitalia. Null mice had micropenis and no scrotal testes
compared with +/+ or +/- littermates. E, Gross appearance of testes
and accessory glands. Testes were small and the size of epididymides,
seminal vesicles, and prostate dramatically decreased in -/- mice
compared with +/+ or +/- littermates. Prostate could not even be
recognized by the naked eye and was difficult to dissect from the
underlying tissue. F, Wet weight of testes and accessory glands. *,
P < 0.01; and **, P < 0.001,
compared with corresponding tissues from +/+ and +/- littermates. The
data presented were the means ± SEs of measurements
on three animals.
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Null males had abdominal testes and a micropenis with a shorter
anogenital distance at 60 days of age compared with +/+ and +/-
littermates (Fig. 3D
). The size of testes, epididymides, and seminal
vesicles dramatically decreased in -/- animals compared with +/+ and
+/- littermates (Fig. 3E
). The prostates were so hypoplastic that they
were barely recognizable even under a dissection microscope and were
difficult to dissect from the underlying tissue. The wet weights of
testes, epididymides, and seminal vesicles dramatically decreased in
-/- animals compared with +/+ and +/- littermates (Fig. 3F
).
Gonadal and Nongonadal Morphology in LH/hCG Receptor Knockout
Animals
Ovaries of -/- animals contained preantral and antral but no
preovulatory follicles or corpora lutea, suggesting a follicular arrest
beyond the antral stage (Fig. 4
, e and
f). The thickness of all uterine layers decreased, and only a few
glands were present in endometrium (Fig. 4
, k and l). The treatment of
these animals with PMSG resulted in a greater number of small antral
follicles with no other obvious changes in either ovaries or in the
reproductive tract (data not shown).

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Figure 4. Histology of Ovaries (af) and Uteri (gl)
Preantral and antral follicles, but not preovulatory follicles or
corpora lutea, were present in ovaries (e and f) and very few glands
present in the endometrium (k and l) of -/- mice. Wild-type and +/-
mice were indistinguishable in their ovarian (a, b, c, and d) and
uterine (g, h, I, and j) morphology. CL, Corpus luteum; FL, follicle;
G, endometrial gland. Magnification: panels a, c, e, g, i, and k, 60x;
panels b, d, f, h, j, and l, 300x.
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Wild-type and +/- animals were indistinguishable in their ovarian
(Fig. 4
, panels a and b vs. panels c and d) and uterine
(Fig. 4
, panels g and h vs. panels i and j) morphology. The
presence of corpora lutea indicates the normal progression of
follicular growth to ovulation. The uterine wall was thick with normal
morphology with numerous glands in endometrium. While both these animal
groups were fertile, -/- females were infertile.
The low power histological examination revealed a marked reduction in
the seminiferous tubule diameters and a drastic decrease in the Leydig
cell number, which were hypotropic in -/- animals compared with +/+
and +/- animals (Fig. 5
, panel e
vs. panels a and c). High-power pictures demonstrate the
arrest of spermatogenesis beyond round spermatid stage in
homozygous animals (Fig. 5
, panel f vs. panels b and
d).

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Figure 5. Histology of Testes
Null mice contained fewer hypotrophic Leydig cells, and sperm were
absent in seminiferous tubules compared with +/+ and +/- littermates,
in which sperm were indistinguishable. Arrowheads
indicate Leydig cells. Magnification: panels a, c, and e, 60x; panels
b, d, and f, 300x.
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Epididymal, seminal vesicle, and prostate histology in -/- animals
was consistent with marked hypoplasia compared with +/+ and +/-
littermates (Fig. 6
, panels c, f, i, and
l vs. panels a, b, d, e, g, h, j, and k). Not only were the
epididymal tubule diameters much smaller, they also were completely
devoid of sperm, and seminal vesicles and prostates contained fewer
acini in -/- animals. The morphology of testes and secondary sex
organs was similar between +/+ and +/- males. While both these animal
groups were fertile, -/- males were infertile.

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Figure 6. Histology of Epididymides (ac), Seminal Vesicles
(df), and Prostates (gl)
All the accessory organs of -/- mice were hypoplastic compared with
+/+ and +/- littermates in which accessory organs were
indistinguishable. Magnification: panels af, g, i, and k, 60x; h, j,
and l, 300x.
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Serum Hormone Levels
Serum LH levels were dramatically elevated and FSH levels were
moderately elevated in -/- female and male animals compared with +/+
and +/- littermates (Fig. 7
, A, B, E,
and F). Estradiol and progesterone levels decreased, but were not
totally suppressed, in -/- females compared with +/+ and +/-
littermates (Fig. 7
, C and D). Testosterone levels were dramatically
decreased and estradiol levels were moderately increased in -/- males
compared with +/+ littermates (Fig. 7
, G and H). Hormone levels in +/-
were similar to +/+ animals except for a modest elevation in FSH and a
decrease in testosterone levels (Fig. 7
, G and H).

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Figure 7. Serum Hormone Levels of Gonadotropins and Steroid
Hormones
a and f, [-/-] vs. [+/+] and [+/-] at
P < 0.0001; b and g, [+/-] vs.
[+/+] at P < 0.05; c and h, [-/-]
vs. [+/+] and [+/-] at P <
0.05; d and j, [-/-] vs. [+/+] at
P < 0.05; e and k, [-/-] vs.
[+/+] and [+/-] at P < 0.05; i, [+/-]
vs. [+/+] at P < 0.01. The data
presented were the means ± SEs of measurements on
four to eight animals in each group.
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Effect of Steroid Hormone Replacement Therapy
Twenty one-day estradiol and progesterone replacement therapy of
30-day-old -/- females resulted in normal vaginal development (not
shown) but had no effect on ovarian morphology (Fig. 8
, a and b). The uterus became thicker;
however, the number of endometrial glands remained low (Fig. 8
, d
vs. c). The animals were still infertile.

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Figure 8. Histology of Ovaries (a and b), Uteri (c and d),
Testes (e and f), Epididymides (g and h), Seminal Vesicles (i and j),
and Prostates (k and l) in Estrogen and Progesterone (b and d),
Testosterone (f, h, j, and l) Replaced and Placebo-Treated (a, c, e, g,
i, and k) -/- Mice
Hormone replacement therapy had improved uterine, testicular,
epididymal, seminal vesicle, and prostate morphology. However, the
number of endometrial glands remained low and the ovarian morphology
was unaffected. There was a resumption of spermatogenesis, but the
sperm numbers remained low. Germ cells in both testes and epididymis
showed high pyknosis. Magnification: a and b, 60x; c, d, and gl,
300x; e and f, 800x.
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Twenty one-day testosterone replacement therapy of 30-day-old -/-
males resulted in a scrotal descent of testes and growth of the penis
(not shown). In addition, therapy caused an enlargement of seminiferous
tubule diameters with resumption of spermatogenesis (Fig. 8
, f
vs. e) but failed to restore Leydig cell number or improve
hypotrophy. Therapy also improved the morphology of epididymides
with enlargement of tubules, which contain spermatozoa, and seminal
vesicles and prostate showed increased size and number of acini (Fig. 8
, panels h, j, and l vs. panels g, I, and k). The size and
weight of these organs compared with that of +/+ animals appeared to
have been restored, yet the animals remained infertile.
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DISCUSSION
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LH is important for follicular maturation, ovulation, and
increasing the synthesis of ovarian steroid hormones (11, 12, 13, 14, 23, 29, 30, 31, 32). Males require LH for Leydig cell proliferation and maturation
and increasing the synthesis of testosterone, which promotes
spermatogenesis and also regulates the function of accessory sex organs
(22, 24, 25, 26, 33, 34).
Numerous reports from several laboratories demonstrated that LH is also
capable of regulating functions of the reproductive tract, brain, skin,
mammary glands, adrenal zona reticularis, urinary bladder, cells of
immune system, etc. (10, 15, 16, 17, 18, 19, 20, 21, 22). Since LH controls the functions of
many tissues, it would be important to have either LH
receptor-deficient or LH-deficient animal models to further advance
current understanding on the total LH actions in the body. The LH
receptor deficiency would be a better model than the LH-deficient
animal because of the possibility that there might be similar
molecule(s) in vivo that might interact with the receptors.
In any case, LH deficiency through targeted disruption of LH-ß
subunit gene has not been done.
In the past, investigators have used the approach of LH deprivation to
investigate the importance of its actions in reproduction (24, 35, 36, 37, 38, 39).
Although this approach gave valuable insights, questions remained on
how much residual LH is left in circulation to act.
We have inactivated LH receptors by gene targeting in embryonic stem
cells. Gonads of these animals had no receptor transcripts or receptor
protein. Despite the fact that LH regulates a wide variety of body
functions, there was no evidence of increased mortality during
embryogenesis or during pre- or postnatal life through at least day 60.
This should not be a surprise considering the fact that lethality
generally results from inactivation of genes that are required for
formation of vital body organs. LH receptor disruption, however, may
have affected the quality of life (i.e. behavior changes,
feeding problems, weakened immune system, skeletal changes,
etc.).
LH receptor gene inactivation had no effect on FSH receptor mRNA levels
in either ovaries or testes, PR mRNA levels in ovaries, and AR mRNA
levels in testes. ER
and StAR mRNA levels decreased and ERß mRNA
levels increased in both ovaries and testes, indicating that, either
directly or indirectly, LH maintains ER
and StAR and inhibits ERß
mRNA levels. It is unlikely that gonadal cell type changes could
account for all mRNA changes in -/- animals. Reciprocal ER changes in
LH receptor knockout animals is consistent with a concept that the two
ERs may have different roles in regulating gonadal functions (40, 41).
Neither cholesterol side-chain cleavage enzyme nor CYP17 mRNA levels
were determined in ovaries or testes of -/- animals.
The LH receptor knockout females were about 50% heavier with a lot of
visceral fat at 60 days of age compared with +/+ and +/- littermates.
Males also showed a lot of visceral fat at a later age (
120 days),
but were lighter which could be due to decreased muscle mass and bone
density. Whether these and other changes in LH receptor knockout
animals would affect their health and longevity is not known. ERKO and
ARKO females were also reported to be heavier with an accumulation of
body fat (40, 42). However, it is not known whether the distribution
pattern or type of fat would be the same between these and LH receptor
knockout animals.
Animals with only one functioning LH receptor allele (+/-) were
indistinguishable from +/+ littermates except for a slight reduction in
testicular 125I-hCG binding, moderate reduction
in serum testosterone levels, and a modest increase in serum FSH levels
in both sexes. Obviously, none of these changes have affected their
fertility or litter size; however, whether these would be affected as
the animals grow older is not known.
LH receptor knockout animals have normal genitalia except that they
were hypoplastic, suggesting that LH signaling is not required for
early gonadal and reproductive tract differentiation. LH-independent
synthesis of testosterone by early fetal Leydig and Mullerian
inhibitory substance by Sertoli cells may allow their differentiation
(43, 44). After they are formed, LH signaling seems to be required for
their continued development through pre- and postnatal life.
Null mice showed several external and internal phenotypic defects at 60
days of age. For example, female animals are acyclic and had an
ambiguous vaginal opening, and their ovaries and reproductive tract
were smaller and lighter. Ovarian histology indicated an arrest in
folliculogenesis beyond the antral stage, which reaffirms that
follicular growth through this stage is independent of LH signaling.
The reproductive tract was underdeveloped with a decreased thickness of
all uterine layers and few glands in the endometrium.
Null males had a micropenis and abdominal testes. The size and weight
of the testes dramatically decreased. The seminiferous tubules were in
disarray, their diameters decreased, and spermatogenesis was arrested
beyond round spermatid stage. Scarce intertubular connective tissue
contained only a few hypotropic Leydig cells, which appeared to be
fetal type by the lack of 11ß-hydroxysteroid dehydrogenase
immunostaining. This indicates that LH signaling is required for adult,
but not fetal, type Leydig cell development. All accessory sex organs
were small to rudimentary with a dramatic decrease in weight.
Serum LH levels are markedly elevated in -/- animals, which could be
due to a loss of estradiol (female) and testosterone (male) negative
feedback and/or loss of negative LH feedback on its own secretion
through decreased hypothalamic GnRH levels in both sexes (45, 46, 47).
Moderate elevation of serum FSH levels could be due to decreased
gonadal inhibin secretion in both sexes. Continued steroid synthesis in
growing follicles through the antral stage under FSH influence may have
prevented a greater decrease than was noted in estradiol and
progesterone levels. The androgen precursor for this continued
estradiol synthesis may come from LH-independent basal synthesis by
theca with small amounts made by granulosa cells. Serum testosterone
levels were undetectable in -/- females as they were in +/+
littermates. Levels in -/- males, on the other hand, dramatically
decreased, which reflects Leydig cell hypoplasia and hypotrophy. The
low androgen levels seen in these animals could be coming from these
few remaining Leydig cells or from their adrenals. The moderate
increase in estradiol levels in -/- males could be due to elevated
FSH levels driving increased Sertoli cell synthesis and/or decreased
estradiol metabolism (48). It is unlikely, however, that they came from
aromatization in adipose tissue because androgen precursor levels were
not elevated.
We used hormone replacement therapy to determine whether phenotype and
biochemical changes were due to decreased gonadal steroid hormone
levels, which in females were not totally suppressed, or they resulted
from loss of LH signaling. If they were due solely to decreased
steroid-hormone levels, then their restoration should correct them.
Lack of reversal of ovarian morphology is consistent with a concept
that only LH, not estradiol, progesterone or FSH, can stimulate
follicular growth beyond the antral stage and induce ovulation. In
relation to biochemical changes, only ER
and StAR decreases were
reversed, whereas the ERß increase was not reversed, suggesting that
LH signaling was inhibitory and is required to maintain normal ovarian
ERß levels. Since there was no resumption of follicular growth beyond
the antral stage and anovulation, -/- animals placed on estradiol and
progesterone replacement therapy remained infertile.
Testosterone replacement therapy resulted in the testes descent into
the scrotum, suggesting that it is androgen dependent. The penis grew
but it remained small compared with +/+ animals, suggesting that it is
not completely dependent on androgens. The decrease in testicular ER
and StAR and increase in ERß mRNA levels were not reversed by
testosterone replacement therapy, suggesting that LH signaling may be
required for their reversal. Neither hyperplasia nor hypotrophy of
Leydig cell was corrected. The therapy increased the diameter of
seminiferous tubules and the resumption of spermatogenesis. Although
sperm number remained relatively low, they were motile. The morphology
of other accessory sex organs was improved. Despite these changes,
treated -/- males remained infertile even after the length of
testosterone replacement therapy was increased to 42 days. There could
be many reasons (behavioral and ejaculatory problems) for continued
infertility in these animals, which we are now beginning to
investigate.
Anovulatory phenotype was seen not only in LH receptor but also in FSH
receptor (49, 50), FSHß (51), ER
(40, 41), ER
/ERß (41),
aromatase (42), PR (52), COX-2 (53), cyclin D2 (54), p27 (Kipl) (55),
and glycoprotein hormone
-subunit (56) knockout animals. Various
degrees of spermatogenic failure that did or did not affect fertility
have been reported after the disruption or overexpression of several
genes (40, 42, 49, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69). These findings indicate that ovulation and
spermatogenesis are the end results of a series of molecular changes
controlled by a number of different factors, and disruption of any one
of them can result in ovulation and spermatogenic failure.
Targeted disruption of the LH receptor gene resulted in a loss of
receptors from uterus, oviduct, brain, skin, mammary gland, urinary
bladder, etc. If the phenotype and biochemical changes in nongonadal
tissues of LH receptor knockout animals were due solely to decreased
steroid hormone levels, which in the case of females were not totally
suppressed, then their restoration should correct them. We have
obtained data on a few tissues and are in the process of obtaining the
rest on the other tissues. As of now, we found that the vagina was
underdeveloped in -/- mice but became normal after estradiol and
progesterone replacement therapy. This suggests that vaginal
development is ovarian steroid hormone dependent. However, although the
vaginal cytology seemed to be improved, the number of leukocytes
remained markedly low, suggesting that something other than ovarian
steroid hormones is required for this reversal. Whether LH signaling is
the answer is not known. The reversal of uterine morphology, except the
endometrial gland number, suggests that perhaps LH actions may be
required for complete gland restoration. Uterine ERß, but not ER
mRNA, decreased in LH receptor knockout animals. This decrease could
not be reversed by estradiol and progesterone-replacement therapy,
suggesting that LH may also be required for the reversal of ERß
decrease. Thus, we are beginning to get a sense that ovarian steroid
hormones alone may not be adequate to maintain complete phenotype of
nongonadal tissues. Thus, LH receptor knockout animals will be useful
in further investigating the importance of LH signaling in nongonadal
tissues.
Since LH and FSH are indispensable for gonadal regulation, it is of
interest to compare the consequences of inactivating one vs.
the other receptor gene. It turns out that the main similarity was that
females in both cases were infertile (49, 57). The main difference was
that FSH receptor knockout males had partial spermatogenic failure,
reduced fertility, and normal to reduced development of accessory sex
organs compared with complete spermatogenic failure, infertility and
hypoplastic accessory sex organs in LH receptor knockout males (49, 57, 69). The other notable differences were that the folliculogenesis was
arrested at the preantral stage and ovarian ER
and ERß were
unaffected in FSH receptor knockout animals compared with arrest beyond
the antral stage, a decrease in ovarian ER
, and an increase in ERß
mRNA levels in LH receptor knockout animals (49, 50, 57). These
findings suggest that, while FSH and LH signaling are equally important
for female fertility, LH signaling is more important in maintaining
male fertility through initiating spermatogenesis via androgens. A
dramatic decrease in testosterone levels in LH receptor-inactivated
animals compared with FSH receptor-inactivated animals may explain male
phenotype differences.
Experimental LH receptor inactivation and naturally occurring
inactivating human LH receptor mutations should be expected to give
rise to similar phenotypes. Thus, it is of interest to compare
similarities and differences between them. The shared similarity was
infertility in both sexes (70, 71, 72, 73, 74). The phenotype of internal male
genitalia and histology was quite similar in both cases. The
differences were that affected men have external female genitalia
(except breast development), which were not seen in LH receptor
knockout animals. Affected women presumably had normal external and
internal genitalia, both of which were dramatically underdeveloped in
LH receptor knockout mice. Some of these differences could reflect
developmental differences between mice and humans, while others may
reflect partial instead of complete inactivating human mutations (75, 76).
In summary, LH/hCG receptor gene disruption resulted in female and male
infertility with several external and internal phenotypic defects and
gene expression changes in gonads. Infertility could not be reversed by
hormone replacement therapy even though some of the phenotypic defects
were corrected. The LH receptor-disrupted animals are anticipated to
increase our current understanding of gonadal and nongonadal actions of
LH and hCG.
 |
MATERIALS AND METHODS
|
---|
Construction of the Targeting Vector
Based on the published sequence of 2.2 kbp 5'-region of the
mouse LH/hCG receptor gene (77), a 199-bp DNA fragment (131329 bp)
was designed as a probe to screen a mouse ES-129/OLA P1 genomic DNA
library (Genome Systems, St. Louis, MO). The DNA fragment
containing about 10.1 kbp of the 5'-region, 0.5 kbp of the first exon,
and 2.5 kbp of the first intron sequence have been identified. A pPNT
vector (a gift from Dr. Colin Funk, University of Pennsylvania,
Philadelphia, PA) that contains PGKneomycin (PGKneo) and PGKthymdine
kinase (PGKtk) cassettes, separated and flanked by a number of unique
cloning sites, was used to construct the LH/hCG receptor targeting
vector. PGKneo served to insert DNA fragment for disruption of the
LH/hCG receptor gene and also as a positive selection marker. PGKtk was
included for negative selection marker. A 4.6-kbp fragment of the 5'-
region of the LH/hCG receptor gene was subcloned downstream of PGKneo
cassette and a 2.5-kbp fragment containing only 21 bp of exon 1 and a
part of intron 1 of the LH/hCG receptor gene was inserted between
PGKneo and PGKtk gene cassettes. The orientation of PGKneo and PGKtk
gene cassettes was in the opposite direction of the LH/hCG receptor
gene to avoid any possibility of fake activation of the LH/hCG receptor
gene by PGKneo promoter (Fig. 1
). The predicted targeted recombination
event would replace the 5'-flanking region and most of the exon 1
sequence (4 kbp) of the LH/hCG receptor gene with PGKneo sequence (1.8
kbp).
Generation of LH/hCG Receptor Knockout Mice
The targeting vector was linearized at a unique NotI
site that lies outside the homologous sequences. Thirty micrograms of
linearized DNA were electroporated into 1 x
107 of 129/SVJ embryonic stem (ES) cells
(Genome Systems)at 185 V and 500 mF (BTX ECM-600,
Genetronics, Inc., San Diego, CA). Then, ES cells were grown on a
feeder layer of mitomycin-inactivated mouse embryonic fibroblasts
(Genome Systems) and selected in medium containing 350
µg/ml G418 and 2 mM ganciclovir (a generous
gift from Roche Products Ltd, Welwyn Garden City,
Herfordshire, UK). A total of 264 doubly-resistant ES clones was
genotyped by digesting 10 µg of their genomic DNA with
StuI or SphI and Southern blotting with
[32P]-labeled 5'- (A probe), 3'- (B probe), or
neomycin probes (Fig. 1
). Southern blotting with all three probes
confirmed the disruption of LH/hCG receptor gene and the integration of
neomycin gene into host genome.
Chimeric mice with ES cells carrying the disrupted LH/hCG receptor
allele were generated by microinjection of 3.5-day-old C57BL/6
blastocysts, which were transferred into the uteri of pseudopregnant
recipient mice (University of Cincinnati Gene Targeted Mouse Service
Center, Cincinnati, OH). Chimeric animals were mated with C57BL/6
(Taconic Farms, Inc., Germantown, NY) or 129/SVJ
(Charles River Laboratories, Inc. Wilmington, MA)
partners. Agouti offspring were genotyped by digesting 10 µg of their
tail genomic DNA with StuI and/or SphI and
Southern blotting with [32P]-labeled A or B
probes. Although the results of DNA digestion with StuI and
hybridization with A probe are presented, predicted DNA fragments
(wild-type, 9.4 kbp, and targeted allele, 1.2 kbp) were obtained when
digested with SphI and Southern blotted with B probe. The
use of neomycin probe on SphI-digested DNA showed no
wild-type fragment and targeted allele fragments of 5.9 and 1.2
kbp.
Male and female +/- animals were crossed to obtain -/- mice, who
were also genotyped by Southern blotting with the same probes. The
crossing of +/- animals had no obvious effect on litter size. Among
the litter, approximately 25% were +/+, 50% were +/-, and 25% were
-/-, indicating that there was no increased intrauterine mortality
among -/- fetuses.
All animals were housed in rooms with 12-h light, 12-h dark cycles with
free access to food and water. Estrous cycles were monitored by daily
vaginal smears and +/- and +/+ animals were killed on the day of
proestrus. The developmental status of external and internal genitalia
was determined at 60 days of age. At least 48 +/+, 48 +/-, and 79
-/- animals were used in these studies. Included in the count of
-/- animals were 20 animals that were placed on hormone replacement
therapy.
RT-PCR
A nonquantitative procedure was used for detection of LH/hCG
receptor mRNA, and a semiquantitative procedure was used for mRNAs of
the others (78). For both, total RNA was isolated using a single-step
acid guanidinium thiocyanate-chloroform extraction method. Master Amp
RT-PCR kits (Epicentre Technologies Corp., Madison, WI) were
used for cDNA synthesis and amplification. Briefly, cDNA was
synthesized from 5 µg RNA using 3'-primer of mouse LH/hCG receptor
cDNA (765 to 785 bp, 5'-AGTGAGTAGGATGACGTGGCG-3'). The cDNA was then
amplified for 40 cycles with 5'-LH/hCG receptor primer (347 to 367 bp,
5'-CCTGCTATACATTGAACCCGG-3'). The RNA was also amplified using
housekeeping gene GAPDH primers to verify the integrity of isolated RNA
samples.
For semiquantitative RT-PCR, 2 µg of total RNA were reverse
transcribed into cDNA with oligo dT primer and AMV reverse
transcriptase (Invitrogen, San Diego, CA). The cDNA was
then coamplified with ß-actin primers,
[32P]-dCTP and one of the following primer sets
(top strand is 5'-primer and bottom strand is 3'-primer). All PCR
primers were designed from published mouse sequences using a Designer
PCR computer program (Research Genetics, Inc., Huntsville,
AL) and synthesized by Operon Technologies Inc. (Alameda,
CA). Optimal conditions and a PCR cycle number for each set of primers
were predetermined to ensure that coamplification was within linear
range.
ER
: 5'-CACATTCCTTCCTTCCGTCTTA-3' and
5'-TCGGGGTAGTTGAACACAGTG-3'
ERß: 5'-ACCAGGACTTACTGCTGAATGC-3' and
5'-GTAGGAATGCGAAACGAGTTGA-3'
PR: 5'-TCTACCCGCCATACCTCAACT-3' and 5'-CTTACGACCTCCAAGGACCAT-3'
Androgen receptor (AR): 5'-ATGGGACCTTGGATGGAGAA-3' and
5'-CCCTGCTTCATAACATTTCCG-3'
FSH receptor (FSHR): 5'-TTGTGGTCATCTGTGGTTGCT-3' and
5'-GCCAAACTTGCTCATCAGGA-3'
StAR: 5'-GGAACCCAAATGTCAAGGAG-3' and 5' and
CTGAGCAGCCAAGTGAGTTTAG-3'
PCR products were resolved by electrophoresis in agarose
(nonquantitative) or polyacrylamide gels (semiquantitative), and both
ethidium bromide staining (nonquantitative) and autoradiography
(semiquantitative) identified the bands. Intensities of the bands were
quantified by a Z-gel Scanning System (Zaxis Inc., Hudson, OH) and
expressed as ratios with ß-actin.
Immunocytochemistry
This procedure was performed by an avidin-biotin
immunoperoxidase method (45, 79). The polyclonal LH/hCG receptor
antibody raised against a synthetic N terminus amino acid sequence of
1538, kindly provided by Dr. Patrick Roche, at the Mayo Clinic
(Rochester, MN), was used at 1:500 dilution. Preabsorption of the
receptor antibody with excess receptor peptide and omission or
substitution of unabsorbed receptor antibody with normal rabbit serum
were used for immunostaining controls.
Ligand Binding Assays
Unlabeled hCG (CR-127 from the National Hormone and Pituitary
Program supported by NIDDK, NICHHD, and US Department of Agriculture)
was radioiodinated by the lactoperoxidase technique (80). One hundred
microgram protein aliquots of gonadal homogenates were incubated for
2 h at 38 C with 1 x 105 cpm
[125I]hCG in the presence or absence of 5
µg/ml of unlabeled hCG. Receptor-bound
[125I]hCG was separated from free hormone by
centrifugation for 20 min at 5,000 x g, and the
radioactivity in the pellets was counted.
Hormone Assays
Mice were anesthetized with ether and exsanguinated by cardiac
puncture. Sera were separated and stored at -80 C until assayed. LH,
FSH, estradiol, progesterone, and testosterone levels were measured in
duplicate using immuno- or RIA kits [LH and FSH kits from
Amersham Pharmacia Biotech (Arlington Heights, IL) and
estradiol, progesterone, and testosterone kits from Diagnostic Products, (Los Angeles, CA)]. All assays were performed
according to procedures provided by the manufacturers. The inter- and
intraassay coefficients of variations were within 515%.
Histological Analysis
Tissues were fixed in 10% formalin overnight and embedded in
paraffin. Then, 5 µm thick tissue sections were cut and stained with
hematoxylin and eosin and examined under bright-field microscopy and
photographed.
Hormone Replacement Therapy
Twenty-one day release 3-mm pellets (Innovative Research of America, Sarasota, FL) were implanted subcutaneously at the
back of the neck of 30-day-old -/- animals with a precision trocar.
The pellets integrate principles of diffusion, erosion, and
concentration gradient, resulting in a biodegradable matrix that
effectively and continuously releases the hormones. Testosterone (5 mg)
pellets were used in males, and pellets containing 0.1 mg
17ß-estradiol and 5 mg progesterone were used in females. Placebo
pellets were implanted into control -/- animals. The animals were
killed at the end of 21 days and tissues were removed. The serum
hormone levels were in the physiological range in hormone-replaced
animals.
Statistical Analysis
The data presented are the means ± SEs. ANOVA
and Duncans multiple range tests were used for determination of
significant difference (81).
 |
ACKNOWLEDGMENTS
|
---|
We thank Mr. Fred Carman for measuring the hormone levels.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. C. V. Rao, Department of Obstetrics/Gynecology, 438 MDR Building, University of Louisville Health Sciences Center, Louisville, KY 40292. E-mail:
cvrao001{at}gwise.louisville.edu, web site:
This work was supported by grants from NIH, Kentucky
EPScoR, and the University of Louisville.
Portions of this work were presented at the 82nd Annual Meeting
of The Endocrine Society in Toronto, Ontario, Canada, June 2024,
2000; The Society for the Study of Reproduction Annual Meeting
in Madison, Wisconsin, July 1518, 2000; and the 11th International
Congress of Endocrinology Meeting in Sydney, Australia, October
29-November 2, 2000.
Received for publication July 31, 2000.
Revision received October 12, 2000.
Accepted for publication October 13, 2000.
 |
REFERENCES
|
---|
-
Pierce JG, Parsons TF 1981 Glycoprotein hormones:
structure and function. Annu Rev Biochem 50:466495
-
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield
RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human
chorionic gonadotropin. Nature 369:455461[CrossRef][Medline]
-
McFarland KC, Sprengel R, Phillips HS, Köhler M,
Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor; an unusual member of the
G-protein-coupled receptor family. Science 245:494499[Medline]
-
Loosfelt H, Misrahi M, Atger M, Salesse R, Vu MT, Thi HL,
Jolivet A, Guiochon-Mantell A, Sar S, Jallal B, Garnier J, Milgrom E 1989 Cloning and sequencing of porcine LH/hCG receptor cDNA: variants
lacking transmembrane domain. Science 245:525528[Medline]
-
Dufau ML 1998 The luteinizing hormone receptor. Annu Rev
Physiol 60:461496[CrossRef][Medline]
-
Tsai-Morris CH, Buczko E, Wang W, Xie X-Z, Dufau ML 1991 Structure organization of the rat luteinizing hormone (LH) receptor
gene. J Biol Chem 266:1135511359[Abstract/Free Full Text]
-
Koo YB, Ji I, Slaughter R, Ji T 1991 Structure of the
luteinizing hormone receptor gene and multiple exons of the coding
sequence. Endocrinology 128:22972308[Abstract]
-
Koo YB, Ji I, Ji TH 1994 Characterization of different sizes
of rat luteinizing hormone/chorionic gonadotropin receptor messenger
ribonucleic acids. Endocrinology 134:1926[Abstract]
-
Wang H, Ascoli M, Segaloff DL 1991 Multiple luteinizing
hormone/chorionic gonadotropin receptor messenger ribonucleic acid
transcripts. Endocrinology 129:133138[Abstract]
-
Rao ChV 1996 The beginning of a new era in reproductive
biology and medicine: expression of low levels of functional
luteinizing hormone/human chorionic gonadotropin receptors in
nongonadal tissues. J Physiol Pharmacol 47:4153
-
Richards JS 1980 Maturation of ovarian follicles: actions and
interactions of pituitary and ovarian hormones on follicular cell
differentiation. Physiol Rev 60:5189[Free Full Text]
-
Rao ChV 1982 Receptors for gonadotropins in human ovaries. In:
Muldoon TG, Mahesh VB, Perez-Ballester B (eds) Recent Advances in
Fertility Research. Developments in Reproductive Endocrinology, Part A.
Liss, New York, pp 123135
-
Richards JS 1994 Hormonal control of gene expression in the
ovary. Endocr Rev 15:725751[Medline]
-
Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN 1998 Molecular mechanisms of ovulation and luteinization. Mol Cell
Endocrinol 145:4754[CrossRef][Medline]
-
Ziecik AJ, Derecka-Reszka K, Rzucidlo SJ 1992 Extragonadal
gonadotropin receptors, their distribution and function. J Physiol
Pharmacol 43:3349
-
Rodway MR, Rao ChV 1995 A novel perspective on the role of
human chorionic gonadotropin during pregnancy and in gestational
trophoblastic disease. Early Preg Biol Med 1:176187
-
Rao ChV, Sanfilippo JS 1997 New understanding in the
biochemistry of implantation: potential direct roles of luteinizing
hormone and chorionic gonadotropin. Endocrinologist 7:107111
-
Rao ChV 1997 Potential novel roles of luteinizing hormone and
human chorionic gonadotropin during early pregnancy in women. Early
Preg Biol Medicine 3:19
-
Rao ChV 1998 Novel concepts in neuroendocrine regulation of
reproductive tract functions. In: Bazer FW (ed) The Endocrinology of
Pregnancy. Humana Press, Totowa, NJ, chapt. 5, pp 125144
-
Rao ChV 1999 A paradigm shift on the targets of luteinizing
hormone/human chorionic gonadotropin actions in the body. J Bellevue
Obstet Gynecol Soc 15:2632
-
Lei ZM, Rao ChV 2000 Endocrinology of trophoblast tissue. In:
Becker K, Rebar R (eds) Principle and Practice of Endocrinology and
Metabolism, ed 3. Lippincott Williams & Wilkins, Philadelphia, in
press
-
Lei ZM, Rao ChV 2000 Direct luteinizing hormone regulation of
the male reproductive tract. In: Countinho AM, Spinola P (eds) Current
Knowledge in Reproductive Medicine. Elsevier Science B.V., Amsterdam,
The Netherlands, in press
-
Robker RL, Richards JS 1998 Hormonal control of the cell cycle
in ovarian cells: proliferation versus differentiation. Biol Reprod 59:476482[Free Full Text]
-
Mendis-Handagama SMLC 1997 Luteinizing hormone on Leydig cell
structure and function. Histol Histopathol 12:869882[Medline]
-
Schlatt S, Meinhardt A, Nieschlag E 1997 Paracrine regulation
of cellular interactions in the testis: factors in search of a
function. J Endocrinol 137:107117
-
Hikim APS, Swerdloff RS 1999 Hormonal and genetic control of
germ cell apoptosis in the testis. J Reprod Fertil 4:3847
-
Tao Y-X, Lei ZM, Rao ChV 1995 Novel expression of luteinizing
hormone (LH)/human chorionic gonadotropin (hCG) receptor gene in rat
epididymis. Biol Reprod 53[Suppl 1]:Abstract 338
-
Eblen A, Bao S, Lei ZM, Sanfilippo JS, Rao ChV 1998 Human
sperm contains luteinizing hormone and chorionic gonadotropin
receptors. J Soc Gynecol Invest 5[Suppl 1]:Abstract 278
-
Catt KJ, Harwood JP, Clayton RN, Davies TF, Chan V, Katikineni
M, Nozu K, Dufau ML 1980 Regulation of peptide hormone receptors and
gonadal steroidogenesis. Recent Prog Horm Res 36:557622[Medline]
-
Misrahi M, Beau I, Meduri G, Bouvattier C, Atger M, Loosfelt
H, Ghinea N, Hai MV, Bougnéres PF, Milgrom E 1998 Gonadotropin
receptors and the control of gonadal steroidogenesis: physiology and
pathology. Bailliéres Clin Endocrinol Metab 12:3566[Medline]
-
Filicori M 1999 The role of luteinizing hormone in
folliculogenesis and ovulation induction. Fertil Steril 71:405414[CrossRef][Medline]
-
Andersen CY, Ziebe S, Guoliang X, Byskov AG 1999 Requirements
for human chorionic gonadotropin and recombinant human luteinizing
hormone for follicular development and maturation. J Assist Reprod
Genet 16:425430[CrossRef][Medline]
-
Williams-Ashman HG 1983 Regulatory feature of seminal vesicle
development and function. Curr Top Cell Regul 22:201275[Medline]
-
Cunha GR, Donjacour AA, Cooke PS, Mee S, Bigsby RM, Higgins
SJ, Sugimura Y 1987 The endocrinology and developmental biology of the
prostate. Endocr Rev 8:338362[Medline]
-
Moudgal NR, Rao AJ, Manekjee R, Muralidhar K, Venkataramiah M,
Sheela Rani CS 1974 Gonadotropins and their antibodies. Recent Prog
Horm Res 30:4777[Medline]
-
Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing
hormone deprivation in situ of steroidogenesis of rat Leydig
cells purified by a multi-step procedure. Biol Reprod 36:769783[Abstract]
-
Keeney DS, Ewing LL 1990 Effects of hypophysectomy and
alterations in spermatogenic function on Leydig cell volume, number,
and proliferation in adult rats. J Androl 11:367378[Abstract/Free Full Text]
-
Keeney DS, Mendis-Handagama SMLC, Zirkin BR, Ewing LL 1988 Effect of long-term deprivation of luteinizing hormone on Leydig cell
volume, Leydig cell number and steroidogenic capacity of the rat
testis. Endocrinology 123:29062915[Abstract]
-
Sriraman V, Rao VS, Sairam MR, Rao AJ 2000 Effect of deprival
of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and
IGF-1. Mol Cell Endocrinol 162:113120[CrossRef][Medline]
-
Couse JF, Korach KS 1999 Estrogen receptor null mice: what
have we learned and where will they lead us? Endocr Rev 20:358417[Abstract/Free Full Text]
-
Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ,
Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking
estrogen receptors
and ß. Science 286:23282331[Abstract/Free Full Text]
-
Fisher CR, Graves KH, Parlow AF, Simpson ER 1998 Characterization of mice deficient in aromatase (ArKO) because of
targeted disruption of the cyp19 gene. Proc Natl Acad Sci
USA 95:69656970[Abstract/Free Full Text]
-
George FW, Catt KJ, Neaves WB, Wilson JD 1978 Studies on the
regulation of testosterone synthesis in the fetal rabbit testis.
Endocrinology 102:665673[Medline]
-
Huhtaniemi I 1994 Fetal testisa very special endocrine
organ. Eur J Endocrinol 130:2531[Medline]
-
Lei ZM, Rao ChV 1994 Novel presence of luteinizing
hormone/human chorionic gonadotropin (hCG) receptors and the
down-regulating action of hCG on gonadotropin releasing hormone gene
expression in immortalized hypothalamic GT17 neurons. Mol Endocrinol 8:11111121[Abstract]
-
Mores N, Krsmanovic L, Catt KJ 1996 Activation of LH receptors
expressed in GnRH neurons stimulates cyclic AMP production and inhibits
pulsatile neuropeptide release. Endocrinology 137:57315734[Abstract]
-
Lei ZM, Rao ChV 1997 Cis-acting elements and
trans-acting proteins in the transcriptional inhibition of
gonadotropin releasing hormone gene by human chorionic gonadotropin in
immortalized hypothalamic GT17 neurons. J Biol Chem 272:1436514371[Abstract/Free Full Text]
-
Carreau S, Genissel C, Bilinska B, Levallet J 1999 Topical
review: sources of oestrogen in the testis and reproductive tract of
the male. Int J Androl 22:211233[CrossRef][Medline]
-
Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur
M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH)
signaling in vivo: targeted disruption of the FSH receptor
leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad
Sci USA 95:1361213617[Abstract/Free Full Text]
-
Danilovich N, Sairam MR, Babu PS, Xing W, Gerdes M 1999 Estrogen deficiency, obesity, and skeletal abnormalities in
follitropin (FSH) receptor knockout (FORKO) female mice. Program of the
Annual Meeting of the American Society for Reproductive
Medicine, (Abstract 0075)
-
Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating
hormone is required for ovarian follicle maturation but not male
fertility. Nat Genet 15:201204[Medline]
-
Lydon JP, De Mayo F, Funk CR, Mani SK, Hughes AR, Montgomery
CA, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit reproductive abnormalities. Genes Dev 9:22662278[Abstract]
-
Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos
JM, Dey SK 1997 Multiple female reproductive failures in
cyclooxygenase-2 deficient mice. Cell 91:197208[Medline]
-
Sicinski P, Donaher PL, Parker SB, Geng Y, Gardner H, Park MY,
Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig J, Bronson RT,
Elledge SJ, Weinberg RA 1996 Cyclin D2 is an FSH-responsive gene
involved in gonadal cell proliferation and oncogenesis. Nature 384:470474[CrossRef][Medline]
-
Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E,
Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM 1996 A syndrome of multiorgan hyperplasia with features of gigantism,
tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85:733744[Medline]
-
Kendall SK, Samuelson LC, Saunders TL, Wood RI, Camper SA 1995 Targeted disruption of the pituitary glycoprotein hormone alpha-subunit
produces hypogonadal and hypothyroid mice. Genes Dev 9:20072019[Abstract]
-
Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG,
Charlton HM 2000 The effect of a null mutation in the
follicle-stimulating hormone receptor gene on mouse reproduction.
Endocrinology 141:17951803[Abstract/Free Full Text]
-
Knudson CM, Tung KSK, Tourtellotte WG, Brow GAJ, Korsmeyer SJ 1995 Bax-deficient mice with lymphoid hyperplasia and male germ cell
death. Science 270:9699[Abstract]
-
Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G 1996 Severe impairment of spermatogenesis in mice lacking the CREM
gene. Nature 380:162165[CrossRef][Medline]
-
Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N,
Poorman-Allen P, Goulding EH, Eddy EM 1996 Targeted gene disruption of
Hsp 702 results in failed meiosis, germ cell apoptosis, and male
infertility. Proc Natl Acad Sci USA 93:32643268[Abstract/Free Full Text]
-
Nantel F, Monaco L, Foulkes NS, Masqulier D, LeMeur M,
Henriksen K, Dierich A, Pavinen M, Sassone-Corsi P 1996 Spermiogenesis
deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 380:159162[CrossRef][Medline]
-
Roest HP, Klaveren J, de Wit J, van Gurp, Koken MHM, Vermey M,
Roijen JH, Hoogerbrugge JW, Vreeburg JTM, Baarends WM, Bootsma D,
Grootegoed JA, Hoijmakers JHJ 1996 Inactivation of the HR6B
ubiquitin-conjugating DNA repair enzyme in mice causes male sterility
associated with chromatin modification. Cell 86:799810[Medline]
-
Rugglu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders
P, Dorin J, Cooke HJ 1997 The mouse Dazla gene enclodes a
cytoplasmic protein essential for gametogenesis. Nature 389:7377[CrossRef][Medline]
-
Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth
J, Hatton KS, Reddy EP 1997 Arrest of spermatogenesis and defective
breast development in mice lacking A-myb. Nature 386:713717[CrossRef][Medline]
-
Ross AJ, Waymire KG, Moss JE, Parlow AF, Skinner MK, Russell
LD, MacGregor GR 1998 Testicular degeneration in B 131
lw-deficient mice. Nat Genet 18:251256[CrossRef][Medline]
-
Meng I, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG,
Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG,
Westphal H, Saarma M, Sariola H 2000 Regulation of cell fate decision
of undifferentiated spermatogonia by GDNF. Science 287:14891493[Abstract/Free Full Text]
-
Wong RW-C, Kwan RW-P, Mak PH-S, Mak KK-L, Sham M-H, Chan S-Y 2000 Overexpression of epidermal growth factor induced
hypospermatogenesis in transgenic mice. J Biol Chem 275:1829718301[Abstract/Free Full Text]
-
Gnessi L, Basciani S, Mariani S, Arizzi M, Spera G, Wang C,
Bondjers C, Karlsson L, Betsholtz C 2000 Leydig cell loss and
spermatogenic arrest in platelet-derived growth factor
(PDGF)-A-deficient mice. J Cell Biol 149:10191025[Abstract/Free Full Text]
-
Krishnamurthy H, Danilovich N, Morales CR, Sairam MR 2000 Qualitative and quantitative decline in spermatogenesis of the
follicle-stimulating hormone receptor knockout (FORKO) mouse. Biol
Reprod 62:11461159[Abstract/Free Full Text]
-
Kremer H, Kraaij R, Toledo SPA, Post M, Fridman JB, Hayashida
CY, van Reen M, Milgrom E, Ropers H-H, Mariman E, Themmen APN, Brunner
HG 1995 Male pseudohermaphroditism due to a homozygous missense
mutation of the luteinizing hormone receptor gene. Nat Genet 8:160164
-
Toledo SPA, Brunner HG, Kraaij R, Post M, Dahia PLM, Hayashida
CY, Kremer H, Themmen APN 1996 An inactivating mutation of the
luteinizing hormone receptor causes amenorrhea in a 46,XX female.
J Clin Endocrinol Metab 81:38503854[Abstract]
-
Themmen APN, Martens JWM, Brunner HG 1998 Activating and
inactivating mutations in LH receptors. Mol Cell Endocrinol 145:137142[CrossRef][Medline]
-
Stavrou SS, Zhu Y-S, Cai L-Q, Katz MD, Herrera C,
Defillo-Ricart M, Imperato-McGinley J 1998 A novel mutation of the
human luteinizing hormone receptor in 46XY and 46 XX sisters. J
Clin Endocrinol Metab 83:20912098[Abstract/Free Full Text]
-
Arnhold IJP, Latronico AC, Batista MC, Mendonca BB 1999 Menstrual disorders and infertility caused by inactivating mutations of
the luteinizing hormone receptor gene. Fertil Steril 71:597601[CrossRef][Medline]
-
Laue LL, Wu S-M, Kudo M, Bourdony CJ, Cutler Jr GB, Hsueh AJW,
Chan W-Y 1996 Compound heterozygous mutations of the luteinizing
hormone receptor gene in Leydig cell hypoplasia. Mol Endocrinol 10:987997[Abstract]
-
Misrahi M, Meduri G, Pissard S, Bouvattier C, Beau I, Loosfelt
H, Jolivet A, Rappaport R, Milgrom E, Bougneres P 1997 Comparison of
immunocytochemical and molecular features with the phenotype in a case
of incomplete male pseudohermaphroditism associated with a mutation of
the luteinizing hormone receptor. J Clin Endocrinol Metab 82:21592165[Abstract/Free Full Text]
-
Huhtaniemi IT, Eskola V, Pakarnen P, Matikainen T, Sprengel R 1992 The murine luteinizing hormone and follicle-stimulating hormone
receptor genes: transcription initiation sites, putative promoter
sequences and promoter activity. Mol Cell Endocrinol 88:5566[CrossRef][Medline]
-
Zhang W, Lei ZM, Rao ChV 1999 Immortalized hippocampal cells
contain functional luteinizing hormone/human chorionic gonadotropin
receptors. Life Sci 64:20832098[CrossRef]
-
Lei ZM, Rao ChV, Kornyei JL, Licht P, Hiatt ES 1993 Novel
expression of human chorionic gonadotropin/luteinizing hormone receptor
gene in brain. Endocrinology 132:22622270[Abstract]
-
Rao ChV, Griffin LP, Carman FR 1977 Gonadotropin receptors in
human corpora lutea of the menstrual cycle and pregnancy. Am J
Obstet Gynecol 128:146153[Medline]
-
Steele RGD 1960 Principles and Procedures of Statistics, with
Special Reference to the Biological Sciences. McGraw-Hill, New
York