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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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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). 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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). 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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, 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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, 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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, 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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, 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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, 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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
15–38, 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 5–15%.
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 Duncan’s multiple range tests were used for determination of
significant difference (81).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 20–24, 2000; The Society for the Study of Reproduction Annual Meeting in Madison, Wisconsin, July 15–18, 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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 P. Tai, K. Shiraishi, and M. Ascoli Activation of the Lutropin/Choriogonadotropin Receptor Inhibits Apoptosis of Immature Leydig Cells in Primary Culture Endocrinology, August 1, 2009; 150(8): 3766 - 3773. [Abstract] [Full Text] [PDF]
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 U. Kolthur-Seetharam, K. Teerds, D. G. d. Rooij, O. Wendling, M. McBurney, P. Sassone-Corsi, and I. Davidson The Histone Deacetylase SIRT1 Controls Male Fertility in Mice Through Regulation of Hypothalamic-Pituitary Gonadotropin Signaling Biol Reprod, February 1, 2009; 80(2): 384 - 391. [Abstract] [Full Text] [PDF]
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 B. D. Looyenga and G. D. Hammer Genetic Removal of Smad3 from Inhibin-Null Mice Attenuates Tumor Progression by Uncoupling Extracellular Mitogenic Signals from the Cell Cycle Machinery Mol. Endocrinol., October 1, 2007; 21(10): 2440 - 2457. [Abstract] [Full Text] [PDF]
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R. Lapatto, J. C. Pallais, D. Zhang, Y.-M. Chan, A. Mahan, F. Cerrato, W. W. Le, G. E. Hoffman, and S. B. Seminara Kiss1 / Mice Exhibit More Variable Hypogonadism than Gpr54 / Mice Endocrinology, October 1, 2007; 148(10): 4927 - 4936. [Abstract] [Full Text] [PDF]
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K. Shiraishi and M. Ascoli Lutropin/Choriogonadotropin Stimulate the Proliferation of Primary Cultures of Rat Leydig Cells through a Pathway that Involves Activation of the Extracellularly Regulated Kinase 1/2 Cascade Endocrinology, July 1, 2007; 148(7): 3214 - 3225. [Abstract] [Full Text] [PDF]
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M. A. Lawson, R. Tsutsumi, H. Zhang, I. Talukdar, B. K. Butler, S. J. Santos, P. L. Mellon, and N. J. G. Webster Pulse Sensitivity of the Luteinizing Hormone {beta} Promoter Is Determined by a Negative Feedback Loop Involving Early Growth Response-1 and Ngfi-A Binding Protein 1 and 2 Mol. Endocrinol., May 1, 2007; 21(5): 1175 - 1191. [Abstract] [Full Text] [PDF]
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 F. J. Diaz, K. Wigglesworth, and J. J. Eppig Oocytes determine cumulus cell lineage in mouse ovarian follicles J. Cell Sci., April 15, 2007; 120(8): 1330 - 1340. [Abstract] [Full Text] [PDF]
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B.K. Campbell, N.R. Kendall, and D.T. Baird The Effect of the Presence and Pattern of Luteinizing Hormone Stimulation on Ovulatory Follicle Development in Sheep Biol Reprod, April 1, 2007; 76(4): 719 - 727. [Abstract] [Full Text] [PDF]
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H. Zhang, J. S. Bailey, D. Coss, B. Lin, R. Tsutsumi, M. A. Lawson, P. L. Mellon, and N. J. G. Webster Activin Modulates the Transcriptional Response of LssT2 Cells to Gonadotropin-Releasing Hormone and Alters Cellular Proliferation Mol. Endocrinol., November 1, 2006; 20(11): 2909 - 2930. [Abstract] [Full Text] [PDF]
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P. Cameo, M. Szmidt, Z. Strakova, P. Mavrogianis, K. L. Sharpe-Timms, and A. T. Fazleabas Decidualization Regulates the Expression of the Endometrial Chorionic Gonadotropin Receptor in the Primate Biol Reprod, November 1, 2006; 75(5): 681 - 689. [Abstract] [Full Text] [PDF]
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 K. L. Matthiesson, R. I. McLachlan, L. O'Donnell, M. Frydenberg, D. M. Robertson, P. G. Stanton, and S. J. Meachem The Relative Roles of Follicle-Stimulating Hormone and Luteinizing Hormone in Maintaining Spermatogonial Maturation and Spermiation in Normal Men J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3962 - 3969. [Abstract] [Full Text] [PDF]
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 K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
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 R. K. Naz and R. Sellamuthu Receptors in Spermatozoa: Are They Real? J Androl, September 1, 2006; 27(5): 627 - 636. [Full Text] [PDF]
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K. L. Matthiesson and R. I. McLachlan Male hormonal contraception: concept proven, product in sight? Hum. Reprod. Update, July 1, 2006; 12(4): 463 - 482. [Abstract] [Full Text] [PDF]
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K. Shiraishi and M. Ascoli Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Stimulates Tyrosine Kinase Cascades that Activate Ras and the Extracellular Signal Regulated Kinases (ERK1/2) Endocrinology, July 1, 2006; 147(7): 3419 - 3427. [Abstract] [Full Text] [PDF]
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S. A. Pangas, X. Li, E. J. Robertson, and M. M. Matzuk Premature Luteinization and Cumulus Cell Defects in Ovarian-Specific Smad4 Knockout Mice Mol. Endocrinol., June 1, 2006; 20(6): 1406 - 1422. [Abstract] [Full Text] [PDF]
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F.P. Yuan, D.X. Lin, C.V. Rao, and Z.M. Lei Cryptorchidism in LhrKO animals and the effect of testosterone-replacement therapy Hum. Reprod., April 1, 2006; 21(4): 936 - 942. [Abstract] [Full Text] [PDF]
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A. H. Vesper, L. T. Raetzman, and S. A. Camper Role of Prophet of Pit1 (PROP1) in Gonadotrope Differentiation and Puberty Endocrinology, April 1, 2006; 147(4): 1654 - 1663. [Abstract] [Full Text] [PDF]
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 C M Allan, Y Wang, M Jimenez, B Marshan, J Spaliviero, P Illingworth, and D J Handelsman Follicle-stimulating hormone increases primordial follicle reserve in mature female hypogonadal mice. J. Endocrinol., March 1, 2006; 188(3): 549 - 557. [Abstract] [Full Text] [PDF]
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 R.-S. Ge, Q. Dong, C. M. Sottas, V. Papadopoulos, B. R. Zirkin, and M. P. Hardy In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development PNAS, February 21, 2006; 103(8): 2719 - 2724. [Abstract] [Full Text] [PDF]
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 A. Roy and M. M Matzuk Deconstructing mammalian reproduction: using knockouts to define fertility pathways Reproduction, February 1, 2006; 131(2): 207 - 219. [Abstract] [Full Text] [PDF]
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R. Toyama, T. Fujimori, Y. Nabeshima, Y. Itoh, Y. Tsuji, R. Y. Osamura, and Y.-I. Nabeshima Impaired Regulation of Gonadotropins Leads to the Atrophy of the Female Reproductive System in klotho-Deficient Mice Endocrinology, January 1, 2006; 147(1): 120 - 129. [Abstract] [Full Text] [PDF]
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M. P.A. van Bragt, N. Ciliberti, W. L. Stanford, D. G. de Rooij, and A. M.M. van Pelt LY6A/E (SCA-1) Expression in the Mouse Testis Biol Reprod, October 1, 2005; 73(4): 634 - 638. [Abstract] [Full Text] [PDF]
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 Y. Zhang, N. Fatima, and M. L. Dufau Coordinated Changes in DNA Methylation and Histone Modifications Regulate Silencing/Derepression of Luteinizing Hormone Receptor Gene Transcription Mol. Cell. Biol., September 15, 2005; 25(18): 7929 - 7939. [Abstract] [Full Text] [PDF]
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T R. Kumar What have we learned about gonadotropin function from gonadotropin subunit and receptor knockout mice? Reproduction, September 1, 2005; 130(3): 293 - 302. [Abstract] [Full Text] [PDF]
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M. Ascoli Learning New Tricks from an Old Dog: The Processing of the Intracellular Precursor of the Luteinizing Hormone Receptor (LHR) into the Mature Cell-Surface LHR Is a Regulated Process Endocrinology, August 1, 2005; 146(8): 3221 - 3223. [Full Text] [PDF]
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P. M. Apaja, J. T. Aatsinki, H. J. Rajaniemi, and U. E. Petaja-Repo Expression of the Mature Luteinizing Hormone Receptor in Rodent Urogenital and Adrenal Tissues Is Developmentally Regulated at a Posttranslational Level Endocrinology, August 1, 2005; 146(8): 3224 - 3232. [Abstract] [Full Text] [PDF]
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D.X. Lin, Z.M. Lei, and Ch.V. Rao Dependence of Uterine Cyclooxygenase2 Expression on Luteinizing Hormone Signaling Biol Reprod, August 1, 2005; 73(2): 256 - 260. [Abstract] [Full Text] [PDF]
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R.-S. Ge, Q. Dong, C. M. Sottas, H. Chen, B. R. Zirkin, and M. P. Hardy Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis Biol Reprod, June 1, 2005; 72(6): 1405 - 1415. [Abstract] [Full Text] [PDF]
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T. Pakarainen, F.-P. Zhang, S. Makela, M. Poutanen, and I. Huhtaniemi Testosterone Replacement Therapy Induces Spermatogenesis and Partially Restores Fertility in Luteinizing Hormone Receptor Knockout Mice Endocrinology, February 1, 2005; 146(2): 596 - 606. [Abstract] [Full Text] [PDF]
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X. Ma, Y. Dong, M. M. Matzuk, and T. R. Kumar Targeted disruption of luteinizing hormone {beta}-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility PNAS, December 7, 2004; 101(49): 17294 - 17299. [Abstract] [Full Text] [PDF]
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M. J. Fields and M. Shemesh Extragonadal Luteinizing Hormone Receptors in the Reproductive Tract of Domestic Animals Biol Reprod, November 1, 2004; 71(5): 1412 - 1418. [Abstract] [Full Text] [PDF]
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Z.M. Lei, S. Mishra, P. Ponnuru, X. Li, Z.W. Yang, and Ch.V. Rao Testicular Phenotype in Luteinizing Hormone Receptor Knockout Animals and the Effect of Testosterone Replacement Therapy Biol Reprod, November 1, 2004; 71(5): 1605 - 1613. [Abstract] [Full Text] [PDF]
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J. F. Couse, M. M. Yates, R. Sanford, A. Nyska, J. H. Nilson, and K. S. Korach Formation of Cystic Ovarian Follicles Associated with Elevated Luteinizing Hormone Requires Estrogen Receptor-{beta} Endocrinology, October 1, 2004; 145(10): 4693 - 4702. [Abstract] [Full Text] [PDF]
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K. C. Lo, Z. Lei, Ch. V. Rao, J. Beck, and D. J. Lamb De Novo Testosterone Production in Luteinizing Hormone Receptor Knockout Mice after Transplantation of Leydig Stem Cells Endocrinology, September 1, 2004; 145(9): 4011 - 4015. [Abstract] [Full Text] [PDF]
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Ch.V. Rao, X.L. Zhou, and Z.M. Lei Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Human Adrenal Cortical H295R Cells Biol Reprod, August 1, 2004; 71(2): 579 - 587. [Abstract] [Full Text] [PDF]
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M Myers, K L Britt, N G M Wreford, F J P Ebling, and J B Kerr Methods for quantifying follicular numbers within the mouse ovary Reproduction, May 1, 2004; 127(5): 569 - 580. [Abstract] [Full Text] [PDF]
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C. M. Allan, A. Garcia, J. Spaliviero, F.-P. Zhang, M. Jimenez, I. Huhtaniemi, and D. J. Handelsman Complete Sertoli Cell Proliferation Induced by Follicle-Stimulating Hormone (FSH) Independently of Luteinizing Hormone Activity: Evidence from Genetic Models of Isolated FSH Action Endocrinology, April 1, 2004; 145(4): 1587 - 1593. [Abstract] [Full Text] [PDF]
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G. Wang and M. P. Hardy Development of Leydig Cells in the Insulin-Like Growth Factor-I (IGF-I) Knockout Mouse: Effects of IGF-I Replacement and Gonadotropic Stimulation Biol Reprod, March 1, 2004; 70(3): 632 - 639. [Abstract] [Full Text] [PDF]
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G.-M. Wang, P. J. O'Shaughnessy, C. Chubb, B. Robaire, and M. P. Hardy Effects of Insulin-Like Growth Factor I on Steroidogenic Enzyme Expression Levels in Mouse Leydig Cells Endocrinology, November 1, 2003; 144(11): 5058 - 5064. [Abstract] [Full Text] [PDF]
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S. B. Rulli, P. Ahtiainen, S. Makela, J. Toppari, M. Poutanen, and I. Huhtaniemi Elevated Steroidogenesis, Defective Reproductive Organs, and Infertility in Transgenic Male Mice Overexpressing Human Chorionic Gonadotropin Endocrinology, November 1, 2003; 144(11): 4980 - 4990. [Abstract] [Full Text] [PDF]
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A. S. Calikoglu Adrenocorticotropic Hormone, a New Player in the Control of Testicular Steroidogenesis Endocrinology, August 1, 2003; 144(8): 3277 - 3278. [Full Text] [PDF]
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P. J. O'Shaughnessy, L. M. Fleming, G. Jackson, U. Hochgeschwender, P. Reed, and P. J. Baker Adrenocorticotropic Hormone Directly Stimulates Testosterone Production by the Fetal and Neonatal Mouse Testis Endocrinology, August 1, 2003; 144(8): 3279 - 3284. [Abstract] [Full Text] [PDF]
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M. R. Maduro, K. C. Lo, W. W. Chuang, and D. J. Lamb Genes and Male Infertility: What Can Go Wrong? J Androl, July 1, 2003; 24(4): 485 - 493. [Full Text] [PDF]
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J. Gromoll, J. Wistuba, N. Terwort, M. Godmann, T. Muller, and M. Simoni A New Subclass of the Luteinizing Hormone/Chorionic Gonadotropin Receptor Lacking Exon 10 Messenger RNA in the New World Monkey (Platyrrhini) Lineage Biol Reprod, July 1, 2003; 69(1): 75 - 80. [Abstract] [Full Text] [PDF]
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M. M. Matzuk, F. J. DeMayo, L. A. Hadsell, and T. R. Kumar Overexpression of Human Chorionic Gonadotropin Causes Multiple Reproductive Defects in Transgenic Mice Biol Reprod, July 1, 2003; 69(1): 338 - 346. [Abstract] [Full Text] [PDF]
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K. Tajima, A. Dantes, Z. Yao, K. Sorokina, F. Kotsuji, R. Seger, and A. Amsterdam Down-Regulation of Steroidogenic Response to Gonadotropins in Human and Rat Preovulatory Granulosa Cells Involves Mitogen-Activated Protein Kinase Activation and Modulation of DAX-1 and Steroidogenic Factor-1 J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2288 - 2299. [Abstract] [Full Text] [PDF]
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Z.M. Lei, W. Zou, S. Mishra, X. Li, and Ch. V. Rao Epididymal Phenotype in Luteinizing Hormone Receptor Knockout Animals and Its Response to Testosterone Replacement Therapy Biol Reprod, March 1, 2003; 68(3): 888 - 895. [Abstract] [Full Text] [PDF]
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M. Haywood, J. Spaliviero, M. Jimemez, N. J. C. King, D. J. Handelsman, and C. M. Allan Sertoli and Germ Cell Development in Hypogonadal (hpg) Mice Expressing Transgenic Follicle-Stimulating Hormone Alone or in Combination with Testosterone Endocrinology, February 1, 2003; 144(2): 509 - 517. [Abstract] [Full Text] [PDF]
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P. J. Baker, P. Pakarinen, I. T. Huhtaniemi, M. H. Abel, H. M. Charlton, T. R. Kumar, and P. J. O'Shaughnessy Failure of Normal Leydig Cell Development in Follicle-Stimulating Hormone (FSH) Receptor-Deficient Mice, But Not FSH{beta}-Deficient Mice: Role for Constitutive FSH Receptor Activity Endocrinology, January 1, 2003; 144(1): 138 - 145. [Abstract] [Full Text] [PDF]
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P. Pakarinen, S. Kimura, F. El-Gehani, L. J. Pelliniemi, and I. Huhtaniemi Pituitary Hormones Are Not Required for Sexual Differentiation of Male Mice: Phenotype of the T/ebp/Nkx2.1 Null Mutant Mice Endocrinology, November 1, 2002; 143(11): 4477 - 4482. [Abstract] [Full Text] [PDF]
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M. Haywood, N. Tymchenko, J. Spaliviero, A. Koch, M. Jimenez, J. Gromoll, M. Simoni, V. Nordhoff, D. J. Handelsman, and C. M. Allan An Activated Human Follicle-Stimulating Hormone (FSH) Receptor Stimulates FSH-Like Activity in Gonadotropin-Deficient Transgenic Mice Mol. Endocrinol., November 1, 2002; 16(11): 2582 - 2591. [Abstract] [Full Text] [PDF]
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S. B. Rulli, A. Kuorelahti, O. Karaer, L. J. Pelliniemi, M. Poutanen, and I. Huhtaniemi Reproductive Disturbances, Pituitary Lactotrope Adenomas, and Mammary Gland Tumors in Transgenic Female Mice Producing High Levels of Human Chorionic Gonadotropin Endocrinology, October 1, 2002; 143(10): 4084 - 4095. [Abstract] [Full Text] [PDF]
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K. H. Burns and M. M. Matzuk Minireview: Genetic Models for the Study of Gonadotropin Actions Endocrinology, August 1, 2002; 143(8): 2823 - 2835. [Abstract] [Full Text] [PDF]
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M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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T. Hirakawa, C. Galet, and M. Ascoli MA-10 Cells Transfected with the Human Lutropin/Choriogonadotropin Receptor (hLHR): A Novel Experimental Paradigm to Study the Functional Properties of the hLHR Endocrinology, March 1, 2002; 143(3): 1026 - 1035. [Abstract] [Full Text] [PDF]
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 P. R. Manna, L. Joshi, V. N. Reinhold, M. L. Aubert, N. Suganuma, K. Pettersson, and I. T. Huhtaniemi Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone Hum. Mol. Genet., February 1, 2002; 11(3): 301 - 315. [Abstract] [Full Text] [PDF]
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 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
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 E. A. Stewart Gonadotropins and the Uterus: Is There a Gonad-Independent Pathway? Reproductive Sciences, December 1, 2001; 8(6): 319 - 326. [Abstract] [PDF]
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I. A. Hughes Minireview: Sex Differentiation Endocrinology, August 1, 2001; 142(8): 3281 - 3287. [Abstract] [Full Text] [PDF]
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A. Eblen, S. Bao, Z. M. Lei, S. T. Nakajima, and C. V. Rao The Presence of Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Human Sperm J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2643 - 2648. [Abstract] [Full Text] [PDF]
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S. B. Seminara and W. F. Crowley Jr. Perspective: The Importance of Genetic Defects in Humans in Elucidating the Complexities of the Hypothalamic-Pituitary-Gonadal Axis Endocrinology, June 1, 2001; 142(6): 2173 - 2177. [Full Text] [PDF]
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I. Huhtaniemi and A. Bartke Perspective: Male Reproduction Endocrinology, June 1, 2001; 142(6): 2178 - 2183. [Full Text] [PDF]
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J. S. Richards Perspective: The Ovarian Follicle--A Perspective in 2001 Endocrinology, June 1, 2001; 142(6): 2184 - 2193. [Full Text] [PDF]
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