University of Washington School of Medicine, Department of Genome Sciences, Seattle, WA 98195, USA
* Author for correspondence (e-mail: braun{at}u.washington.edu)
Accepted 20 October 2003
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SUMMARY |
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Key words: Androgen receptor, Sertoli, Spermatogenesis, Mouse
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Introduction |
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In both humans and mice, XY individuals carrying a hemizygous null mutation
in the X chromosome-linked Ar gene exhibit complete Androgen
insensitivity syndrome (cAIS), which is characterized by pseudohermaphroditism
and sterility (Online Mendelian Inheritance in Man,
http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=300068).
Although the phenotype of these individuals clearly demonstrates the crucial
requirement for AR in development, the suitability of AIS as a model for
studying the spermatogenic function of Ar is poor. Testicular descent
fails in mice with cAIS (Artfm), and the spermatogenic
phenotype mimics that of cryptorchidism in an otherwise normal male, namely
early meiotic arrest (Lyon and Hawkes,
1970). Thus, it is impossible to distinguish the contribution to
the phenotype of loss of AR function from that resulting from the abdominal
positioning of the testes. Mice homozygous for a mutation in the gonadotrophin
releasing hormone gene (Gnrh), which have dramatically lowered serum
testosterone levels (Singh et al.,
1995
), present a testicular phenotype similar to cAIS
(Cattanach et al., 1977
).
Spermatogenesis in these animals can be qualitatively rescued by androgen
replacement therapy (Singh et al.,
1995
). This occurs in the absence of appreciable levels of the
gonadotrophins LH and follicle stimulating hormone (FSH). Further, FSH alone
fails to significantly rescue spermatogenesis beyond the meiotic stages
(Haywood et al., 2003
;
Singh and Handelsman, 1996
),
suggesting that T and/or DHT is the major hormonal regulator of
spermatogenesis.
This observation is further supported by classic androgen withdrawal
experiments in rats. Removal of androgens from adult rats by hypophysectomy
(Hx) leads to an acute, stage-specific regression of the seminiferous
epithelium (Ghosh et al.,
1991; Russell and Clermont,
1977
). After long-term Hx and elimination of residual testosterone
by flutamide, spermatogenesis rarely proceeds beyond meiosis, with very few
round spermatids observed and elongated spermatids nearly nonexistent
(Franca et al., 1998
). As with
Gnrh-null mice, androgen or LH replacement leads to qualitative
recovery of spermatogenesis in hypophysectomized rats while FSH has little
direct stimulatory effect on spermatogenesis
(El Shennawy et al., 1998
;
Elkington and Blackshaw, 1974
;
Russell and Clermont, 1977
).
Similar results are seen in response to suppression of Gnrh activity
(Szende et al., 1990
), and
destruction of Leydig cells with the Leydig-specific cytotoxin EDS
(Kerr et al., 1993
;
Sharpe et al., 1990
).
In the mouse testis, AR protein is expressed in the somatic Leydig, myoid
and Sertoli cells (Zhou et al.,
2002). Although expression in Leydig and myoid cells is
continuous, Sertoli cell expression of AR occurs in a stage-dependent fashion.
Interestingly, the stages during which AR expression in Sertoli cells is
highest correspond directly with those most acutely affected by androgen
withdrawal. Sertoli cells are also the only somatic cell type in direct
contact with differentiating germ cells. They provide both physical and
nutritional support for spermatogenesis, which occurs in the intercellular
spaces between Sertoli cells (McGuiness
and Griswold, 1994
). Taken together, these observations have led
to the general belief that Sertoli cells are the primary mediators of AR
regulation of spermatogenesis. Loss of AR activity from Sertoli cells would
thus be responsible for the spermatogenic phenotypes outlined above, primarily
the failure to efficiently complete meiosis.
To crucially examine this hypothesis, we have created a conditional null allele of the Ar gene in mice which allows the selective removal of AR function from Sertoli cells. This conditional Ar allele also proved to be a general hypomorph because of an inhibitory effect of the inserted Pgk-neo cassette on Ar mRNA processing.
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Materials and methods |
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The targeting construct was linearized at the 5' end with NotI and electroporated into the FWB2 mouse embryonic stem cell line, derived in our laboratory from the 129/SvJaeSor strain of mice. Two correctly targeted clones were identified. One of these clones, Ar206, was then injected into C57Bl/6J derived blastocysts to produce chimeric offspring. Males were identified that transmitted the Ar206 Ar allele through the germline. The line of mice derived from these chimeras has been designated Artm1Reb, referred to throughout the text as Arflox(ex1-neo).
To allow Sertoli cell-specific ablation of AR activity, a Cre
recombinase transgene driven by the anti-Mullerian hormone (Amh)
promoter was generated. A 1.5 kb BamHI-KpnI Amh
genomic fragment containing the entire promoter region
(Beau et al., 2001) as well as
the complete first exon and intron 1 was digested with NheI to
linearize the template downstream of the Amh translational initiation
codon. The Amh AUG and the remainder of exon 1 were replaced with a
Myc-tagged Cre cDNA by homologous recombination in yeast. Intron 1 of
the Amh gene was left intact, fused to the 3' end of
Cre. This Amh-cre construct was isolated from the vector by
KpnI-NotI digestion and introduced into FVB/N zygotes by
pronuclear injection. Founder animals were identified by transgene specific
PCR and functionally screened for CRE activity by mating to the R26R
tester strain (Soriano, 1999
)
and their progeny assayed for testis specific ß-galactosidase activity.
Two independent lines expressing Amh-cre specifically in Sertoli
cells were identified and one (line 8815) backcrossed into the 129/SvJaeSor
strain for at least three generations prior to their use in these
experiments.
Southern blots
Genomic DNA (5-10 µg) was digested overnight with EcoRV and separated on
1% agarose gels by electrophoresis for approximately 19 hours at 40 volts (V).
DNAs were transferred to Genescreen nylon membrane (New England Nuclear) by
capillary transfer in 20xSSC. Blots were UV crosslinked and
pre-hybridized 60 minutes at 65°C in [2xSCP, 1% sarkosyl, 0.01%
BSA]. A random primed 32P-labelled DNA probe was generated from a
400 bp Ar exon 1 PCR fragment and hybridized to the membrane
overnight at 65°C in [2xSCP, 1% sarkosyl, 0.01% BSA,
1xDenhardt's solution, 0.1 mg/ml salmon sperm DNA]. The blots were
washed 20 minutes at 65°C in [2xSSC, 1% SDS], 20 minutes at 42
degrees in [0.1xSSC, 0.1% SDS], and 20 minutes at room temperature in
[0.1xSSC, 0.1% SDS], followed by overnight exposure to X-ray film.
Phenotypic analysis of Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y; Amh-cre males
Eight-week-old males of the desired genotypes and wild-type littermate
controls were euthanized by CO2 asphyxiation and body weight
measurements taken. Blood was collected by aortic puncture with a 23-gauge
needle coated with heparin to prevent clotting. Red blood cells were pelleted
by centrifugation and the overlying serum recovered for testosterone, LH, FSH
and estradiol assays. All hormone assays were performed by the University of
Virginia Ligand Core Laboratory.
Testes, epididymides and seminal vesicle were collected from each animal and testis and seminal vesicle weights recorded. A single testis and epididymis from each animal was fixed overnight at 4°C in either Bouin's fixative or neutral-buffered formalin (NBF), rinsed with 70% ethanol, embedded in paraffin wax, cut into 5 µm sections, and mounted on glass slides for histological and immunocytochemical examination. The second epididymis was minced in 1 ml sperm motility buffer (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 30 mM HEPES pH 7.4, 10 mM sodium lactate, 1 mM sodium pyruvate, 20 mg/ml BSA, 25 mM NaHCO3) and sperm allowed to swim out overnight at room temperature. Numbers of epididymal sperm were determined by hemacytometer counts. The remaining testis was subdivided for protein, DNA and RNA isolation. For protein, one-half testis was homogenized in 500 µl buffer A [10 mM HEPES (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT plus PMSF, leupeptin, and pepstatin A as protease inhibitors] followed by addition of 50 µl buffer B [0.3 M HEPES (pH 7.6), 1.4 M KCl, 30 mM MgCl2]. Cellular debris was pelleted by centrifugation and soluble protein extracts stored at -70°C. One-quarter testis was homogenized in 1 ml Trizol reagent (Invitrogen) and total RNA isolated following the manufacturer's instructions. Finally, one-quarter testis was used for DNA purification using the Qiagen DNEasy Tissue kit following the manufacturer's protocol.
Immunocytochemistry and ß-galactosidase detection
Sections from testis and epididymis (5 µm) were deparaffinized and
rehydrated [2x10 minutes Xylenes, 2x5 minutes 100% ethanol,
1x5 minutes 95% ethanol, 1x5 minutes 70% ethanol, 1x5
minutes 50% ethanol, 1x5 minutes phosphate buffered saline (PBS)],
followed by quenching of endogenous peroxidase activity (1x15 minutes
0.5% H2O2). AR immunostaining was performed on NBF fixed
tissues as previously described (Zhou et
al., 2002). MSY4 and PRM2 immunostaining was performed on Bouin's
fixed tissues as previously described
(Giorgini et al., 2002
).
Immunostained sections were counterstained with Hematoxylin and mounted in GVA
mounting solution (Zymed).
For assessment of Amh-cre function, fetal gonads and mesonephroi were dissected from embryonic day 14.5 animals. The dissected tissues, as well as the remainder of the fetus, were fixed 30 minutes in 4% paraformaldehyde at room temperature. All tissues were then rinsed 2x30 minutes in lacZ rinse buffer (200 mM Na2HPO4·7H2O pH 7.3, 2 mM MgCl2, 0.02% Nonidet P-40, 0.01% sodium desoxychoate). The rinsed tissues were incubated overnight at 37°C in lacZ stain [lacZ rinse buffer containing 0.02 M K3Fe(CN)6, 0.02 M K4Fe(CN)6, and 1 mg/ml X-gal] to detect ß-galactosidase activity. After staining, tissues were rinsed in PBS prior to imaging.
Western and northern blots
To determine AR protein levels in testes of control and experimental mice,
20 µg total testis protein was separated on a 10% SDS-PAGE minigel
(BioRad). Proteins were transferred to Protran nitrocellulose membrane
(Schleicher & Schuell) 30 minutes at 20 V on a Trans-Blot SD apparatus
(BioRad). Membranes were blocked 30 minutes in PBS + 5% dry milk prior to
addition of primary antibody. The membrane was cut in half horizontally
through the 84 kilodalton (kDa) molecular weight marker. Proteins greater than
84 kDa were probed with rabbit anti-AR antibody N-20 (sc-816; Santa Cruz
Biotechnology) diluted 1:500. As a loading control, proteins smaller than 84
kDa were probed with mouse anti--tubulin monoclonal antibody (Zymed
Laboratories) diluted 1:3000. Primary antibody incubations were overnight at
4°C in PBS + 5% dry milk. Membranes were washed 2x15 minutes in PBS
+ 0.1% Tween-20. Blots were then incubated with horseradish
peroxidase-conjugated secondary antibody (Bio-Rad), diluted 1:5000 in PBS + 5%
dry milk, for 2-3 hours at room temperature. The membranes were washed as
before followed by detection of peroxidase activity by ECL kit (Amersham) and
exposure to X-ray film.
Pem and Prm1 mRNA levels were determined by northern blot. Total testis RNA (10 µg) was run 2.5 hours at 100 V on a 1.5% formaldehyde-agarose gel. RNA was then transferred overnight to GeneScreen nylon membrane (New England Nuclear) by capillary transfer in 20xSSC. Random primed 32P-labelled probe DNA was generated from cDNAs corresponding to the Pem, Prm1 and ß-actin genes. The RNA was UV crosslinked to the membrane followed by hybridization overnight at 42°C in hybridization buffer (50% formamide, 5xSSC, 50 mM NaPO4 pH 6.5, 250 µg/ml salmon sperm DNA, 1xDenhardt's solution, 0.5% SDS, 6.25% dextran sulfate). The membrane was washed 30 minutes at 65°C in [2xSSC, 1% SDS, 0.1% sodium pyrophosphate] followed by 30 minutes at 65°C in [0.5xSET, 0.1% sodium pyrophosphate]. The membranes were then exposed to a storage phosphor screen and imaged on a Storm 820 phosphorimager (Amersham). Bands corresponding to the RNA of interest were then quantified with ImageQuant software (Amersham). RNA levels for Pem and Prm1 were normalized to the ß-actin loading control to allow between sample comparisons. Membranes were probed sequentially and stripped in boiling 1% SDS between probes.
Statistical analyses
Between group comparisons for all variables were performed by one-way ANOVA
followed by Tukey's post hoc analysis using the Statistical Package for Social
Sciences (SPSS) version 10.0.
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Results |
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In order to specifically remove AR function from Sertoli cells, we created a transgene in which Cre cDNA expression is regulated by the Sertoli cell specific anti-Mullerian hormone promoter (Amh-cre, see Fig. S2 at http://dev.biologists.org/supplemental). To confirm an interaction between Amh-cre and Arflox(ex1-neo)/Y, PCR was performed to detect the expected inversion allele generated by recombination between the loxP sites (Fig. 1D). As anticipated, an inversion specific product is observed in DNA from the testes of Arflox(ex1-neo)/Y; Amh-cre males, but not in the tails of these animals. No product is amplified from tail or testis DNA of Arflox(ex1-neo)/Y males, though an Arflox(ex1-neo) specific PCR product is detected in all samples, confirming the integrity of the DNA.
Morphological and behavioral analysis of Ar mutant phenotypes
Previous work has shown that inclusion of the neomycin phosphotransferase
cassette used in our targeting construct is likely to create a hypomorphic
allele of the gene into which it is inserted
(Meyers et al., 1998).
Therefore we decided to examine the phenotype of
Arflox(ex1-neo)/Y males as potential hypomorphs, along
with the Sertoli cell mutant Arflox(ex1-neo)/Y; Amh-cre
animals. Unlike an Artfm null male,
Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y;
Amh-cre males were indistinguishable from wild-type male littermates by
external examination. In addition, Arflox(ex1-neo)/Y males
produced copulatory plugs when housed with superovulated females, indicating
male sexual behavior is properly imprinted. However, as shown in
Table 1, further
characterization uncovered significant differences not only between mutant
animals and wild type, but also between the mutants themselves.
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Systemic and molecular aspects of reduced Ar function
To further characterize the phenotypes of the mutant animals, measurements
were made of serum gonadotrophin (LH and FSH), testosterone (T) and estradiol
levels. Although no differences were observed between the two mutant classes,
serum LH and T levels were drastically and significantly increased in both
Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y;
Amh-cre males in comparison with Ar+/Y animals
(Table 2). LH levels were
increased 23-fold with a concomitant 40-fold increase in T levels in the
serum. FSH levels were also significantly elevated in mutant animals, though
only approx. twofold relative to wild type. Interestingly, estradiol levels
were unaffected in Ar mutant males.
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Testicular and epididymal abnormalities associated with Ar mutations
Histological analysis of testes and epididymides from Ar mutant
and wild-type males confirmed and extended our previous findings. Overall size
and organization of the epididymis was similar between genotypes. However,
numbers of mature sperm were reduced in the epididymides of
Arflox(ex1-neo)/Y males whereas spermatozoa were
histologically undetectable in Arflox(ex1-neo)/Y; Amh-cre
epididymides (Fig. 3A-C).
Epididymal smooth muscle hyperplasia was also apparent in males of both mutant
genotypes. Additionally, cellular debris was commonly observed in the
epididymal lumen. In particular, large numbers of what appear to be highly
vacuolated round spermatids are observed in the epididymides of
Arflox(ex1-neo)/Y; Amh-cre males.
|
Protein marker analysis by immunocytochemistry provided further insight
into the phenotypes of Arflox(ex1-neo)/Y and
Arflox(ex1-neo)/Y; Amh-cre animals. MSY4, the expression
of which marks mid-stage pachytene spermatocytes through round spermatids
(Davies et al., 2000), is
largely unaffected in the Ar mutants
(Fig. 4A-F). These images also
demonstrate the reduction in numbers of elongating spermatids at stage X-XI in
our Ar mutant animals, with the loss being most severe in
Arflox(ex1-neo)/Y; Amh-cre males. PRM2, which is expressed
in elongated spermatids (Stanker et al.,
1987
), is present in Arflox(ex1-neo)/Y testes,
although the numbers of positive spermatids appears to be reduced
(Fig. 4H). PRM2 positive
spermatids are further reduced in Arflox(ex1-neo)/Y;
Amh-cre testes (Fig. 4I),
consistent with previous observations from sperm counts and epididymal and
testicular histology. As expected, AR protein was detected in the myoid,
Leydig and Sertoli cells of Arflox(ex1-neo)/Y testes, even
in the most severely affected tubules (Fig.
4K). Within the testes of Arflox(ex1-neo)/Y;
Amh-cre males, AR is clearly expressed in myoid and Leydig cells, while
Sertoli cells are AR negative (Fig.
4L). Occasionally, faint AR immunoreactivity is detected within
Sertoli cells (see Fig. S3A at
http://dev.biologists.org/supplemental);
however, this signal is due to expression of a non-functional, N-terminal
peptide generated from an inverted exon 1-neomycin fusion RNA, as described
above. No full-length AR protein is detected within the Sertoli cells of
Arflox(ex1-neo)/Y; Amh-cre males (see Fig. S3C at
http://dev.biologists.org/supplemental).
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Discussion |
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Characterization of Ar hypomorphic males has provided unexpected
insight into the function of AR in prenatal development and spermatogenesis.
Our results clearly demonstrate sensitivity to AR function during sexual
differentiation distinct from the requirement for normal spermatogenesis.
Arflox(ex1-neo)/Y males are morphologically normal in
spite of reduced levels of AR protein, as evidenced by western blot analysis
of testis protein extracts. The dysregulation of serum gonadotrophin levels
also implies decreased AR function in the hypothalamus and pituitary. In
addition, AR activity appears to be reduced based on the decrease in Sertoli
cell transcript levels from the AR-regulated Pem gene. These results
together indicate an overall depression of AR activity in
Arflox(ex1-neo)/Y animals. Not surprisingly, evaluation of
epididymal sperm numbers and testicular histology reveals a severe disruption
of spermatogenesis. Thus, it seems that quantitatively normal spermatogenesis
requires a higher level of AR function than does male sexual differentiation.
This result mimics that found for the mildest forms of androgen-insensitivity
syndrome (AIS) in humans, but to our knowledge this is the first example of
partial AIS (pAIS) in an animal model
(Giwercman et al., 2001;
Hiort et al., 2000
). It is
also worth noting that the Arflox(ex1-neo)/Y phenotype
results from a reduction in wild-type AR protein levels, rather than a
reduction in the activity of individual AR molecules as is nearly always the
case in human pAIS (Androgen Receptor Gene Mutations Database,
http://ww2.mcgill.ca/androgendb/).
As mentioned above, Arflox(ex1-neo)/Y males also
exhibit dysregulation of serum gonadotrophin and testosterone (T) levels.
Interestingly, given their interrelated modulation by GnRH, LH concentration
is more dramatically affected than is that of FSH. This differential effect on
LH and T versus FSH can be explained in the context of known mechanisms of
gonadotrophin regulation. In studies of human gonadotrophin production,
decreased T levels have been shown to increase the frequency and magnitude of
LH and FSH secretion, whereas increases in T levels decrease the frequency of
the GnRH pulse from the hypothalamus, thus suppressing release of LH and FSH
from the pituitary (Matsumoto and Bremner,
1984). In addition, the frequency of the GnRH pulse has been shown
to differentially affect LH and FSH secretion, with high frequency pulses
favoring secretion of LH while less frequent pulses favor FSH
(Wildt et al., 1981
).
Paradoxically, our model is expected to mimic the effect of testosterone
withdrawal in spite of the elevated levels of serum T. Owing to the decrease
in functional AR, the system is effectively experiencing androgen depletion.
Thus, we would expect the frequency of the GnRH pulse to be increased, with
the outcome being preferential secretion of LH over FSH.
FSH secretion is also regulated by factors that do not overlap with the
regulation of LH. Inhibin B, which is produced by Sertoli cells in response to
FSH stimulation, feeds back directly on the pituitary to limit FSH secretion
(Anawalt et al., 1996).
Experiments in Ar-null mice have also shown that T and estradiol act
to suppress serum FSH concentration in the absence of functional androgen
receptor, implying estrogen receptor mediated negative feedback on FSH levels
(Schleicher et al., 1989
).
These mechanisms of FSH regulation, which are expected to be wholly intact in
our model, are thus likely to account for the differential effect on LH and
FSH levels in the Arflox(ex1-neo)/Y hypomorphic male. By
contrast, our results provide strong evidence for a direct and primary role
for AR in the feedback regulation of LH and T levels. This presumably occurs
through direct effects on the hypothalamus and pituitary as well as through
autocrine regulation of T production by the Leydig cells.
The role of AR function during spermatogenesis has been the subject of
intense interest for many years. It has been assumed, reasonably so, that the
major cellular mediator of this regulatory function of AR is the Sertoli cell,
based on its intimate contact with germ cells and stage-specific AR expression
(McGuiness and Griswold, 1994;
Zhou et al., 2002
). Abundant
prior work on the role of testosterone and AR in spermatogenesis has led to
the expectation that Sertoli cell AR function is required for the completion
of meiosis and the transition of spermatocytes to haploid round spermatids. As
previously cited, multiple studies of androgen withdrawal and disruption of AR
activity, either by surgical, chemical or genetic means, have produced similar
results: in the absence of androgens and AR function, spermatogenesis rarely
proceeds beyond meiosis. In all of these model systems, very few round and
even fewer elongated spermatids are observed. This point was made clearly in a
recent study (Haywood et al.,
2003
). In the presence of physiological levels of transgenic FSH,
significant stimulation of Sertoli cell and spermatogonial proliferation was
observed in the testes of Gnrh-null animals. However, only limited
numbers of haploid spermatids were detected whose development was arrested
during elongation. Efficient completion of meiosis and development of mature
spermatozoa required the presence of androgens.
In the current study we have provided evidence for the presence of two AR-sensitive steps during spermatogenesis. The first, which is most acutely sensitive to a reduction in AR function, occurs in the late stages of spermatid differentiation near the time of spermiation. Unexpectedly, the second step, which is sensitive to loss of Sertoli cell AR activity, occurs during the transition from the round to elongating stages of spermiogenesis, and may involve a loss of adhesion of round spermatids to the seminiferous epithelium. Based on our observations, it is possible that a primary role of AR function in Sertoli cells is to regulate spermatid adhesion to the seminiferous epithelium. AR would thus be required for maintenance of adhesion of round spermatids during their differentiation to elongated spermatozoa. Conversely, AR function is then required for the execution of spermiation and release of mature sperm into the tubule lumen. We are currently testing this hypothesis through examination of the cell-to-cell contacts between Sertoli and germ cells.
Sertoli cell Ar expression does not appear to be required for the
completion of meiosis or the differentiation of round spermatids prior to
elongation. However, androgens are clearly required at some level for normal
meiotic progression. It has been recently shown that progestins, which like
the androgens act through a nuclear hormone receptor, are capable of exerting
a non-genomic effect on cell proliferation in the absence of functional
progesterone receptor (Sager et al.,
2003). Thus, we suggest that androgens may still be required in
Sertoli cells for execution of meiosis, but that these hormones are also
capable of acting non-genomically, in the absence of AR function, to promote
spermatogenesis. Alternatively, our results indicate that the search for
localization of the meiotic AR requirement be refocused outside of the
seminiferous tubules. One possible candidate is the peritubular myoid cell,
which expresses AR at all stages of spermatogenesis and is in close proximity
to the spermatogonial stem cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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