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Department of Physiology (F.-P.Z., M.P., I.H.) Institute of
Biomedicine University of Turku FIN-20520, Turku, Finland
Department of Cell and Molecular Biology (J.W.) Karolinska
Institute S-17177 Stockholm, Sweden
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The consequences of inactivation of LH action also remains to be clarified. Only a single man with LHß mutation has been reported (8); he presented with normal sexual differentiation at birth but total lack of postnatal sexual development. No women with such a mutation have yet been described. Neither are there knockout models for LHß or LH receptor (LHR). More is known about consequences of inactivating LHR mutations in man (1). Depending on completeness of the receptor inactivation, men present with pseudohermaphroditism ranging from mild micropenis and hypo-spadias to complete sex reversal. The phenotype in women is milder, including only anovulatory infertility.
In our exploration of the consequences of inactivation of gonadotropin action, we concluded that a knockout mouse model for the LHR would be a logical next step. It is known that LH stimulates Leydig cell differentiation and steroidogenesis in the postnatal testis, but its role in the fetal period is controversial (9). In the ovary, LH stimulates theca cell androgen production, triggers ovulation, and stimulates estrogen and progesterone production of corpus luteum. LH actions in early stages of female development are unlikely, because LHRs appear in the ovary only postnatally (10). In addition, there are recent findings on LHR expression and LH actions in extragonadal organs (11, 12). In addition to the expected phenotype of hypogonadism of the LH receptor knockout (LuRKO) mice, the developmental and possible extragonadal findings were especially hard to predict. Likewise, it was interesting to see, to what extent FSH alone, in the absence of LH action, was able to support gonadal function. We report here the phenotypes of male and female mice with targeted disruption of the LHR gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). After
electroporation of the targeting construct (Fig. 1A) into ES cells, and
after drug selection, 250 surviving colonies were picked and screened
by PCR. Positive clones were further confirmed by Southern blots. Five
of the 250 clones gave a 2.3-kb fragment in PCR analysis, and bands
with expected size of 11 kb for the wild-type (WT) allele and 9 kb for
the targeted allele in Southern blot, indicating that homologous
recombination had occurred (Fig. 1, B and C). Three of these cell lines
(C30, C76 and C101) were injected into blastocysts, yielding nine male
chimeras. The male chimeras were bred with C57BL/6 females, and
germline transmission (F1 offspring) was obtained with three of them.
Heterozygous mice were fertile and viable. Intercrossing the F1
heterozygotes yielded F2 progeny including 43 LHR WT (+/+) (22 female,
21 male), 86 heterozygous (+/-) (32 female, 53 male), and 37
homozygous (-/-) mice (16 female, 21 male), in agreement with the
expected Mendelian mode of inheritance.
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) as those
of WT littermates, and their intraabdominal location adjacent to the
urinary bladder was the same as in WT males. Likewise, the internal
genitalia revealed under stereomicroscopy no difference between (+/+),
(+/-), and (-/-) males (data not shown). From about 3 weeks’
postnatal age the male LuRKO mice were significantly lighter than (+/+)
or (+/-) mice of the same age, and at 45 days this weight difference
reached 30% (results not shown). No clear weight differences were
found between (-/-), (+/-), and (+/+) females up to 7 weeks of age
(data not shown). The LuRKO males could be distinguished from their
normal male littermates by external examination after 30–35 days by
their small penis, short anogenital distance, and underdeveloped
scrotum. At the age of 7 weeks, their testes were very small [(+/+),
94 ± 6.9; (+/-), 91 ± 3.1; (-/-), 17 ± 0.77 mg;
mean ± SEM, n = 8–10] and located in the
abdominal cavity adjacent to the urinary bladder. The accessory
reproductive organs (seminal vesicles, epididymides, and prostates)
were macroscopically invisible (Fig. 2A). Histology of the (-/-)
testes showed that seminiferous tubules were narrower and
spermatogenesis was arrested at the round spermatid stage (Fig. 2). The
numbers and sizes of Leydig cells appeared dramatically decreased
as compared with (+/+) testes (Fig. 2).
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). Ovarian
histology at the age of 7 and 12 weeks showed in (-/-) mice presence
of follicles up to the early antral stage, but no preovulatory
follicles or corpora lutea (Fig. 3). No apparent differences were seen
in the thickness of the theca cell layers surrounding the developing
follicles. The uterine histology showed thinning of all cell layers and
absence of glandular structures (Fig. 3). Comparison of estrous cycles
of the (+/+) and (-/-) mice at the age of 12 weeks showed the cyclic
periodicity with recognizable estrus every 4 days in (+/+) mice, but
not in (-/-) mice.
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Analysis of the Disrupted LHR Gene
The targeting construct was designed so that the pGKneo
insert would replace exon 11, which encodes the transmembrane and
cytoplasmic receptor domains, and part of the 3'-extracellular domain,
thus preventing the formation of full-length functional LHR capable of
anchoring to the plasma membrane and of signal transduction. However,
it is possible that the remaining fragment of the LHR gene could
be transcribed into truncated forms of LHR mRNA. We performed RT-PCR
analysis with different primer pairs on testicular and ovarian RNA from
LuRKO and WT mice. Using primer pair LHRm1 and 2 (specific for the
extracellular domain of LHR) an amplicon of 412 bp was detected from
(+/+), (+/-), and (-/-) testes and ovaries (Fig. 4). Using primer pair LHRm3 and 4
(specific for transmembrane and cytoplasmic domains), a 359-bp band was
found in (+/+) and (+/-) mouse testes and ovaries, but not in those of
(-/-) mice (Fig. 4). Northern hybridization analysis of testicular
RNA by using a cRNA probe specific for extracellular domain of LHR
revealed four major transcripts of LHR mRNA with sizes of 6.9, 2.6,
1.7, and 1.2 kb in the (+/+) and (+/-) mice, but only one band of 1.2
kb in the (-/-) mice. When using the cRNA probe specific for the
transmembrane domain of LHR, two major bands with sizes of 6.9 and 2.6
kb were present in the (+/+) and (+/-) mice, but no hybridization was
observed in the (-/-) samples (data not shown). Hence, the LuRKO
mice do not synthesize any mRNA encoding the full-length functional
LHR.
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).
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). P450
17-OH is a theca cell-specific enzyme and necessary for their androgen
production. In the LHR-deficient ovaries, the P450 17-OH mRNA was
decreased by 12-fold as compared with the expression of WT ovaries
(Fig. 6). These data provide evidence that the LHR gene deletion
profoundly, although not totally, reduces the steroidogenic activities
of the Leydig and theca cells.
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indicated that serum LH levels were dramatically increased in (-/-)
males and females, whereas there were no significant difference of
pituitary LH levels between the (+/+), (+/-), and (-/-) mice. Serum
FSH levels were elevated, and pituitary FSH levels were decreased in
(-/-) males and females as compared with (+/-) and (+/+) males and
females. Serum and testicular testosterone levels of (-/-) males were
significantly decreased (Fig. 8) as
compared with (+/+) and (+/-) males. However, the former levels were
somewhat higher than we detect in castrated mouse circulation (13),
indicating that testicular steroidogenesis was not totally abolished by
LHR inactivation. Likewise, the ovarian estradiol concentration was
significantly decreased in (-/-) female mice compared with (+/+) and
(+/-) females (Fig. 8). Similar suppression of ovarian progesterone
was observed in the LuRKO females (data not shown).
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DISCUSSION |
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Interestingly, the sexual differentiation and gonadal histology of
female and male LuRKO mice were indistinguishable from WT littermates
at birth. In females, this is not surprising, since the early female
sex differentiation is known to be independent of ovarian function
(16), and gonadotropin receptors occur in rodent ovaries only several
days after birth (17). Although fetal testicular testosterone synthesis
is crucial for male sexual differentiation, the LuRKO mice provide, for
the first time, direct evidence that specific elimination of LH action
does not hamper this function. This has been suggested indirectly by
earlier findings on normal intrauterine masculinization of
gonadotropin-deficient hpg (18) and common 
-subunit
knockout (19) mice, and on unmeasurable levels of LH in rat fetal
circulation at the time of the sharpest increase in fetal testicular
testosterone production (20).
The testes of the LuRKO mice weighed about 17 mg, which were
about 18% of that of WT testes, but 5-fold more than those of the
hpg mice (18, 21). This increased weight over hpg
mice can be ascribed to the elevated FSH action in the LuRKO mice,
because this gonadotropin is known to stimulate Sertoli cell
proliferation in the neonatal testis (22, 23, 24). While the
spermatogenesis of hpg and common -subunit knockout mice
proceeds up to the diplotene stage (18, 19), some tubules of the LuRKO
mice show round spermatids, i.e. completion of meiosis. This
is supported by the findings that progression of spermatogenesis from
spermatocytes to spermatids can be stimulated after hypophysectomy by
either FSH or androgen (25). However, both the intraabdominal location
and insufficient testosterone production of the LuRKO testes offset
further progression of spermatogenesis. Interestingly, it is typical
for experimental cryptorchidism with undisturbed Leydig cell
testosterone production (26), including the recently developed Insl3
knockout mouse (27), that they, despite normal androgen levels, lack
postmeiotic germ cells. This raises the possibility that normal
intratesticular testosterone concentration in the abdominal temperature
is deleterious to spermatogenesis, as seems to occur during the
recovery of spermatogenesis after cytotoxic or radiation insults
(28).
Concerning testicular descent, no difference was found at birth in the location of the testes, adjacent to the urinary bladder, between the (-/-) and (+/+) mice. Hence, the lack of LH stimulation in utero did not hamper the first transabdominal phase of testicular descent, known to be dependent on both androgen and Insl3 (29).
The ovaries of the LuRKO mice were reduced in size and histological analysis revealed follicles up to the early antral stage, but no preovulatory follicles or corpora lutea. The ovaries of hpg mice have follicles up to the preantral stage (18), which indicates that FSH action, present in LuRKO mice, has a distinct effect on progression of preantral follicles to the early antral stage. Correspondingly, the lack of preovulatory follicles and corpora lutea indicates that the very last steps of follicular maturation, as well as ovulation, do not occur without LH action. Another intriguing feature of the LuRKO ovaries was the apparent normal thickness of thecal cell layers surrounding the follicles. Hence, although theca cells are a target of LH action, their survival is apparently not dependent on LH, which observation can also be made in hpg ovaries (18). However, theca cell androgen production, to provide substrate for granulosa cell estrogen production, is LH dependent. Therefore, the defective estrogenization of LuRKO females was expected, as demonstrated by their low ovarian estradiol level, delayed sexual maturation, and hypoplastic uteri.
As an indicator of Leydig cell steroidogenic activity (30, 31), we measured the mRNA level of P450scc in the LuRKO testes. The low but detectable level of expression indicates that low constitutive expression of this enzyme is possible in the absence of LH action in the precursor Leydig cells detected in the LuRKO testes. In accordance, the serum testosterone levels in the LuRKO males were slightly higher than measured by us in orchidectomized mice (13). However, the physiological significance of this residual androgen production is unlikely in view of the lack of postnatal sexual development of the LuRKO males. Likewise, low but detectable levels of P450 17-OH mRNA, a marker of theca cell steroidogenesis (32), was detected in the LuRKO ovaries, indicating that this enzyme is also expressed constitutively at low levels. This finding, together with the well developed theca cells and low but detectable estradiol level of the LuRKO ovaries, explains the delayed vaginal opening of the LuRKO females.
Both female and male LuRKO mice represent close phenocopies of the respective human mutation (1). LHR inactivation in males causes pseudohermaphroditism of varying severity. The most severe forms present with female genitals, absence of uterus, low testosterone, and high LH. The milder forms, with partial LHR inactivation, display a broader array of phenotypes ranging from micropenis to hypospadias. The severity of the phenotype has been shown to correlate with the degree of LHR inactivation (1). Although the receptor inactivation in LuRKO mice is total, the male phenotype is less dramatic than in connection with similar human mutations. This indicates that the gonadotropin- independent component of fetal Leydig cell androgen production is more prominent in the mouse than in the human. The presence of chorionic gonadotropin (hCG) in human fetal circulation may explain the difference. Regulation of the critical process of testosterone- dependent male sexual differentiation needs, in addition to pituitary LH, a backup mechanism, which is hCG in the human (33) and testicular paracrine regulation (20) in the rodent.
The female phenotype of LuRKO mice is even closer to that of the inactivating human LHR mutations (1). Affected women have normal primary and secondary sex characteristics, increased gonadotropins, and low estrogen and progesterone production. Likewise, suppressed but not absent estrogen production of the LuRKO mice is reflected by the presence of granulosa cells in their ovaries, delayed vaginal opening, and hypoplastic uteri. Ovarian histology demonstrates follicles at early stages of development, but no preovulatory follicles or corpora lutea, in both women with LHR inactivation and in LuRKO mice. These observations in the human and mouse support the view that LH is essential for normal estrogen production and ovulation, whereas follicular development is initially independent of gonadotropins and, in its final stages, is dependent on FSH and LH.
In conclusion, the LuRKO mouse allows us to identify directly the specific LH- dependent steps of male and female sexual differentiation and adult gonadal functions. It is a close phenocopy of completely inactivating mutations of the human LHR gene, and it provides a valuable tool for experimental studies of pathogenesis of this condition. Although all effects characterized in the present study were concerned with development and function of gonads and sex organs, the LuRKO model also helps us to explore the putative and recently documented extragonadal actions of LH (11, 12), which most notably apply to the tumorigenic effects observed with this hormone (34, 35).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Sequencing confirmed validity of the genomic fragment.
Targeting Vector Construction
The targeting plasmids pKO Scramber913, pKO SelectNeo V800, and
pKO SelectTK V830 were purchased from Lexicon Genetics, Inc. (The
Woodlands, TX). Briefly, the targeting vector contained a 4.2-kb
BamHI-XbaI fragment as the 5'-homology region and
a 2.2-kb EcoRI-BamHI fragment representing the
3'- homology region, a positive selection marker (the PGK-Neo
expression cassette), and an MC1-tk (thymidine kinase) expression
cassette (Fig. 1).
Embryonic Stem (ES) Cell Culture
The ES cell line, AB.2.2-prime ES cells, was purchased
from Lexicon Genetics, Inc. (The Woodlands, TX) and cultured on
neomycin-resistant primary embryonic fibroblast feeder layers
irradiated with 3000 rads. Ten million cells were electroporated (500
µF, 240 V) with 30 µg of linearized targeting construct. After
electroporation, the surviving cells were plated on 100-mm diameter
culture dishes and exposed to G418 (Sigma) at 300 mg/liter
and 1 µM of ganciclovir (Hoffman-LaRoche Inc., Basle, Switzerland) for 9–10 days. Colonies were picked
into 24-well plates and grown for 5–6 days, when about one eighth of a
colony was replated onto 24-well plates for genomic DNA extraction,
with the remainder being frozen at -80 C.
Screening of Targeting Clones
DNA was isolated from each individual clone and screened by PCR,
which produced a 2.3-kb amplicon with a primer pair corresponding to
the 3'-end of pGKneo (Neo1, 5'-GGGCTCTATGGCTTCTGAGGCGGA-3') and to the
flanking 3' end of exon 11 (LHR3, 5'-TCTCAGGGAGGATTTGGGTATGG-3') (Fig. 1A). Correct targeting of the ES cells was further confirmed by
Southern hybridization analysis of HindIII- digested ES
cell genomic DNA and a probe specific for the flanking sequence of
intron 10; the expected band from unmodified LHR was 11 kb in size and
9 kb for the deleted LHR gene.
Mouse Breeding
The targeted ES cells were injected into blastocysts from
C57BL/6J females and implanted into pseudopregnant mothers to proceed
to term. Chimeras were identified by coat color, and males were bred to
C57BL/6J females to test germline transmission. Genotyping of the mice
was carried out by using PCR on genomic DNA with primer pairs for the
WT allele (LHR1, 5'-TCTGGGGATCTTGGAAATGA-3'; LHR2,
5'-CACCTTGACACCTGGAGT-3') and for the targeted allele (Neo1-LHR2) (Fig. 1). Tail DNA from F1 offspring with agouti coat color was screened by
PCR with primer pair Neo1 and LHR2. F2 offspring and the subsequent
generations were screened by PCR using primer pairs for detecting
presence of the targeted LHR gene and the WT gene.
All mice were handled in accordance with the institutional animal care policy of the University of Turku.
Histological Analysis
Testes, ovaries, epididymides, seminal vesicles, uteri, and
pituitary glands were removed, fixed in 4% paraformaldehyde at 4 C for
4–14 h, dehydrated, and embedded in paraffin, and sectioned at 5 µm
thickness. Sections were stained with Harris hematoxylin and eosin (BDH
Ltd., Poole, UK). The reproducibility of all the morphological data was
verified by similar findings in at least three different animals.
RNA Isolation and Analyses
RNA isolation and RT-PCR were carried out as previously reported
(37). RNA was extracted by the single-step method (38). cDNA was
generated by reverse transcriptase (RT) from 2 µg of testicular or
ovarian RNA, using avian myeloblastosis virus (AMV)-RT with
random hexamers (Promega Corp., Madison, WI), in a final
volume of 25 µl. Subsequent PCR analysis was performed on 3 µl of
the cDNA and 0.1 µl of [32P]-CTP (
400
Ci/mmol; Amersham Pharmacia Biotech, Aylesbury, UK ), and
the PCR products were analyzed by electrophoresis on 1.4% agarose
gels. The oligonucleotide primers used for RT-PCR were designed
according to the published cDNA sequences of mouse LHR (39). PCR
amplification with the primer pair LHRm1 and 2 (LHRm1,
5'-TGAACCCGGTGCTTTTACAA-3'; LHRm2, 5'-CGTGGCGATGAGCGTCTGAATG-3'),
specific for the extracellular domain of LHR, yields a 412-bp fragment.
With the primer pair LHRm3 and 4 (LHRm3,
5'-ATCGCCACGTCATCCTACTCACTG-3'; LHRm4, 5'AGCCAAATCAACACCCTAAG-3'),
specific for exon 11 of LHR, a 359- bp amplicon is produced.
Northern Hybridization Analysis
Ten or five micrograms of total RNA from testis and ovary were
resolved on 1.2% formaldehyde denaturing agarose gel and transferred
onto nylon membrane (Hybond-XL, Amersham Pharmacia Biotech). Prehybridization and hybridization were performed as
previously described (37). Briefly, the filters were prehybridized for
at least 4 h at 65 C in a solution containing 50% formamide,
3 x SCC (1 x SCC = 150 mM NaCl and 15
mM sodium citrate, pH 7.0), 5 x Denhardt’ solution,
1% SDS. Hybridization was carried out at 66 C overnight in the same
solution after adding the [32P]-labeled cRNA
probe. After hybridization, the membranes were washed twice with 1
x SCC and 0.1% SDS at 65C for 30 min each time and twice with 0.1 SCC
and 0.1% SDS at 66 C for 30 min each time. The membranes were exposed
to x-ray film (Kodak XAR-5, Eastman Kodak Co., Rochester,
NY) at -70 C for 1–3 days. The molecular sizes of the mRNA species
were estimated by comparison with mobility of the 18S and 28S ribosomal
RNAs. The [32P]-labeled cRNAs were synthesized
using a Riboprobe synthesis II kit (Promega Corp.),
[32P]UTP (Amersham Pharmacia Biotech), and the corresponding cDNA templates. For generation
of P450scc riboprobe, a template composed of a
fragment of rat P450scc cDNA (spanning bp
186–695), subcloned into T vector under T7 RNA polymerase promoter,
was used (40). Antisense cRNA probe for P450 17-OH mRNA analysis was
produced using as template a fragment of mouse P450 17-OH cDNA
(spanning bp 55–616) subcloned into T vector under the T7 RNA
polymerase promoter (41). For the LHR cRNA probes, the cDNAs used as
templates corresponded to bases 441–849 of extracellular domain of rat
LHR cDNA (36), and to bases of 1,002–1,461 of transmembrane domain of
mouse LHR cDNA (36), respectively.
hCG Binding Assay
Testicular LHR binding was measured as previously reported (42, 43). Briefly, a piece of testis tissue was homogenized with an
Ultra-Turrax 18/10 homogenizer in Dulbecco’s PBS + 0.1% BSA (0.5 mg
tissue/ml). Highly purified hCG (NIH CR-125; 13,000 IU/mg) was
radioiodinated using a solid phase lactoperoxidase method.
One-hundred-microliter aliquots of testicular homogenate were incubated
in triplicate at room temperature for 18 h, in the presence of a
saturating concentration (150,000 cpm; 3 ng) of
[125I]iodo-hCG. Nonspecific binding was
assessed in the presence of a 1,000-fold excess of unlabeled hCG
(Pregnyl, Organon, Oss, The Netherlands). The
centrifugation step used to separate bound and free hormone (1000
x g, 30 min at 4 C) precipitates only membrane-bound
receptors (42).
To measure hCG binding to detergent-solubilized (i.e.
membrane-bound and soluble) receptors, the testes were homogenized in
ice-cold buffer A (150 mM NaCl, 20
mM HEPES, pH 7.4) containing 20% glycerol, 1%
Nonidet P-40 (NP-40), and protease inhibitor cocktail
(Sigma), and incubated on ice for 30 min. After
centrifugation at 13,000 rpm for 30 min at 4 C, the supernatant was
used for the ligand binding assay. The binding reaction was carried out
as above, except that the incubation was overnight at 4 C. Free and
bound [125I]iodo-hCG were separated by
precipitating the samples with polyethylene glycol (mol wt 8,000). Each
tube received 0.2 ml of 5 g/liter solution of bovine 
-globulin in
buffer A and 0.5 ml of 30% (wt/vol) polyethylene glycol in buffer A.
After incubation at 4 C for 10 min, the samples were pelleted at 2,800
rpm for 30 min, and supernatants were removed. Pellets were resuspended
in 0.9 ml of buffer A containing 0.1% NP-40 and 20% glycerol. After
addition of 0.5 ml of polyethylene glycol, the tubes were mixed,
incubated at 4C for 10 min, and centrifuged again. The supernatants
were aspirated, and the pellets were counted in a -spectrometer.
Nonspecific binding was determined in these measurements as above.
Hormone Measurements
Serum and pituitary LH and FSH levels were determined by
immunofluorometric assays as earlier described (44, 45).
Intratesticular testosterone and ovarian estradiol and progesterone
were determined by homogenizing one (-/-) testis and a weighed
portion (approximately half) of one (+/+) or (+/-) testis in 0.5 ml
PBS, and a pair of ovaries in 0.2 ml PBS. One hundred microliters of
the gonadal homogenates or serum were extracted twice in 2 ml diethyl
ether and evaporated to dryness overnight in a fume hood. After
reconstitution into PBS, testosterone and progesterone were measured by
standard RIAs. Estradiol level was measured by a DELFIA Estradiol kit
(Wallac, Inc., Turku, Finland) according to the
manufacturer’s instruction. Protein concentrations in homogenates were
measured using the Bradford method (46).
Statistical Analysis
The Statview program (Windows version 4.57; Abacus Concepts
Inc., Berkeley, CA) was used for ANOVA and t tests.
Significance was set as P < 0.05. The values are
presented as mean ± SE.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by grants from the Academy of Finland and The Sigrid Jusélius Foundation.
Received for publication June 7, 2000. Revision received September 25, 2000. Accepted for publication October 2, 2000.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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produces hypogonadal and hypothyroid mice. Genes Dev 9:2007–2019-hydroxylase/C17–20 lyase in
Leydig cells. Biol Reprod 42:399–404[Abstract]
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characterization of ovarian tumors and establishment of
gonadotropin-responsive granulosa cell lines. Mol Endocrinol 10:1667–1677[Abstract]
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S. Zhongyi, P. Rantakari, T. Lamminen, J. Toppari, and M. Poutanen Transgenic Male Mice Expressing Human Hydroxysteroid Dehydrogenase 2 Indicate a Role for the Enzyme Independent of Its Action on Sex Steroids Endocrinology, August 1, 2007; 148(8): 3827 - 3836. [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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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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C A Wilson and D C Davies The control of sexual differentiation of the reproductive system and brain Reproduction, February 1, 2007; 133(2): 331 - 359. [Abstract] [Full Text] [PDF]
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 A. Domanskyi, F.-P. Zhang, M. Nurmio, J. J. Palvimo, J. Toppari, and O. A. Janne Expression and localization of androgen receptor-interacting protein-4 in the testis Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E513 - E522. [Abstract] [Full Text] |
