Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.
ABSTRACT Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.
- [show abstract] [hide abstract]
ABSTRACT: Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.Science 08/1991; 253(5016):202-5. · 31.03 Impact Factor
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ABSTRACT: The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (delta F508-CFTR), reduces Cl- secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO3- transport across cell membranes. HCO3- permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or delta F508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na(+)-free (N-methyl-D-glucamine substitution) solutions (to block Na(+)-dependent pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-free solutions showed Cl- channels with a linear I/V relationship and a conductance of 10.4 +/- 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO3-] on the inside and outside, the Cl-/HCO3- permeability ratio (determined from reversal potentials of I/V curves) was 3.8 +/- 1.0 (mean +/- SEM; n = 9); the ratio of conductances was 3.9 +/- 0.5 (at 150 mM Cl- and 127 mM HCO3-. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO3- through channels that conduct HCO3- about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl- and HCO3- are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO3- secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3- may lead to decreased pH of the luminal fluid.Proceedings of the National Academy of Sciences 07/1994; 91(12):5340-4. · 9.74 Impact Factor
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ABSTRACT: Cystic fibrosis (CF) is a fatal genetic disease caused by abnormalities in fluid and electrolyte transport in exocrine epithelia. Both absorptive and secretory processes are affected by an underlying membrane defect in Cl- permeability. However, the impact of the defect on transport function is tissue specific. Net electrolyte absorption is decreased in the sweat duct, increased in airway epithelia, and not affected in intestine. The defect is expressed in secretion as a consistent failure in most, if not all, exocrine tissues, to beta-adrenergically stimulated and cAMP mediated secretory response. However, the secretory response to cholinergic and Ca2(+)-mediated stimulation is normal in the sweat gland, apparently normal in the airway, but absent in the intestine. The basic defect is not fatal in and of itself, and the imbalance between absorption and secretory functions may be of some selective advantage to heterozygotes in surviving complications of intestinal infections. The inherent defect in transport is probably the primary physiological cause of the ultimately fatal secondary infections in the lungs of CF homozygotes.The FASEB Journal 08/1990; 4(10):2709-17. · 5.70 Impact Factor
Defective CFTR-Dependent CREB Activation Results in
Impaired Spermatogenesis and Azoospermia
Wen Ming Xu1,2., Jing Chen2., Hui Chen2, Rui Ying Diao2,3, Kin Lam Fok2, Jian Da Dong2, Ting Ting Sun2,
Wen Ying Chen2,4, Mei Kuen Yu2, Xiao Hu Zhang2, Lai Ling Tsang2, Ann Lau2, Qi Xian Shi4, Qing Hua Shi5,
Ping Bo Huang6, Hsiao Chang Chan1,2*
1Sichuan University-The Chinese University of Hong Kong Joint Laboratory for Reproductive Medicine, West China Institute of Women and Children’s Health, West China
Second University Hospital, Sichuan University, Chengdu, People’s Republic of China, 2Faculty of Medicine, School of Biomedical Sciences, Epithelial Cell Biology Research
Center, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China, 3Shenzhen Key Lab of Male Reproduction and Genetics, Peking University
Shenzhen Hospital, Shenzhen, People’s Republic of China, 4Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, People’s
Republic of China, 5Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science
and Technology of China, Hefei, People’s Republic of China, 6Department of Biology, Hong Kong University of Science and Technology, Hong Kong, People’s Republic of
Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the
cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR
mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised
spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element
binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO32transport and
the expression of the HCO32sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture
of rat Sertoli cells. Inhibition of CFTR or depletion of HCO32could reduce FSH-stimulated, sAC-dependent cAMP production
and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also
observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in
regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and
azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related
Citation: Xu WM, Chen J, Chen H, Diao RY, Fok KL, et al. (2011) Defective CFTR-Dependent CREB Activation Results in Impaired Spermatogenesis and
Azoospermia. PLoS ONE 6(5): e19120. doi:10.1371/journal.pone.0019120
Editor: Irina Agoulnik, Florida International University, United States of America
Received November 25, 2010; Accepted March 27, 2011; Published May 9, 2011
Copyright: ? 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by the National Natural Science Foundation of China (No. 30
Biosciences in collaboration with Sun Yat-sen University, the National 973 projects (2006CB504002), the Focused Investment Scheme and Li Ka Shing Institute of
Health Sciences of The Chinese University of Hong Kong, and the Morningside Foundation. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
), the South China National Research Center for Integrated
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis
transmembrane conductance regulator (CFTR), a cAMP-activat-
ed anion channel conducting both Cl2and HCO32[1,2]. A
multitude of clinical manifestations are associated with CF, which
include chronic lung inflammation/infection, pancreatic insuffi-
ciency, intestinal obstruction and infertility/subfertility in both
sexes [3,4,5]. However, the exact mechanisms underlying various
CF-related pathological conditions or diseases remain largely
unknown. While previous studies have demonstrated that about
95% of the male CF patients are infertile because of bilateral
absence of the vas deferens (CBAVD) , whether CFTR
mutations may result in other forms of infertility, such as
azoospermia, remains an open question.
CFTR expression has been detected in human [7,8] and rodent
[9,10,11] testes, both in germ cells and Sertoli cells, suggesting its
possible involvement in spermatogenesis. However, controversial
data were obtained when CFTR mutation screening was
performed in infertile patients with defective testicular function,
with some reports indicating the association of CFTR mutations
and defects in sperm production [12,13,14,15], but some others
rejecting the association [16,17,18,19]. Interestingly, in a study
screening a panel of 13 mutations of CFTR in semen specimens
from 127 CF-unrelated healthy males attending infertility clinics,
fourteen (17.5%) of 80 healthy men with infertility due to reduced
sperm quality and two of 21 men (9.5%) with azoospermia carried
one CFTR mutation, while no CFTR mutation was detected in 26
males who had normozoospermia . Another study also shows
that a higher proportion of 5 T allele, a common variation of the
poly(T) in intron 8 of Cftr, which has been associated with low level
of CFTR expression and with susceptibility to non-classical CF
disease patterns, exists in men with severe oligozoospermia [6,20],
suggesting that variation in CFTR may be associated with defects
in spermatogenesis. Despite the accumulating evidence indicating
possible involvement of CFTR in sperm production, the exact role
PLoS ONE | www.plosone.org1May 2011 | Volume 6 | Issue 5 | e19120
of CFTR in spermatogenesis has not been elucidated, and thus,
the question as to whether CFTR mutations may result in
impaired spermatogenesis remains controversial. We undertook
the present study to address this question.
Impaired spermatogenesis in CF mice models
To investigate possibleinvolvementofCFTR inspermatogenesis,
we used a CFTR knockout (Cftrtm1Unc, also referred as S489X) mice.
Since most homozygous S489X CF mice die at a young age or less
frequently available, heterozygous mice were used for quantitative
measurement. Morphological study showed that testis tissue size
than that of wild-type control (Fig. 1A). Daily sperm production
(DSP), which is often used to evaluate the spemiogenesis in the testis
, was significantly reduced in heterozygous S489X CF mice
compared with wide-type control (Fig. 1B). The sperm number
recovered from the epididymis of the heterozygous S489X CF mice
was also significantly lower than that of wide-type control (Fig. 1C).
H&E staining showed slight decrease in diameter of seminiferous
tubules (Fig. 1D,E) and slight cytoplasmic shrinkage of spermato-
cytes and round spermatids (Fig. S1) in S489X CF mice. Realtime
PCR results showed significant reduction of Protamine-2, a specific
marker of spermatid and spermatozoa, in heterozygous and
homozygous S489X CF mice at mRNA level (Fig. 1F). Immuno-
fluorescence results further demonstrated the down-regulation of
Protamine-2 in S489X CF mice at protein level (Fig. 1G). CREM,
a spermatid-specific transcription factor, was also down-regulated in
the homozygous S489X CF mice compared to wild-type as
indicated by Western blot and immunofluorescent staining
respectively (Fig. 1H&J), suggesting defect in spermatogenesis in
CF mice. Interestingly, immunofluorescence result showed that
cAMP-responsive element binding protein (CREB), an important
transcription factor in Sertoli cells during spermatogenesis, was
decreased in the Sertoli cells of homozygous S489X CF mice, as
compared to heterozygotes and wild-type (Fig. 1I). Western blot
result also showed downregulation of phosphorylated and total
CREB in homozygous S489X CF mice compared to wild-type and
heterozygotes (Fig.1K). Thus all the spermatogenesis parameters
examined indicate that spermatogenesis is compromised in CF
mice, confirming a role of CFTR in spermatogenesis.Further more,
down-regulation of phosphorylated and total CREB in CF mice
suggests that defect of CFTR may lead to abnormal regulation of
cAMP-CREB pathway in Sertoli cells, which might be the
molecular basis for the spermatogenesis defect observed in CF.
CFTR is expressed in rodent Sertoli cells and involved in
Hormonal regulation of spermatogenesis, such as by FSH and
testosterone, is primarily targeting Sertoli cells, which are in close
contact with the germ cells and provide paracrine factors that are
important for germ cell development . Given the essential role
of Sertoli cells in spermatogenesis and the reported expression of
CFTR in these cells , we hypothesized that CFTR may play
an important role in spermatogenesis by regulating Sertoli cell
function. In order to test this hypothesis, we established a primary
culture of rat Sertoli cells with .95% purity , since primary
culture of mouse Sertoli cell is not well established. The expression
of CFTR in the cultured Sertoli cells was further confirmed by
RT-PCR (Fig. 2A) and Western blot (Fig. 2B).
A growing body of evidence has demonstrated that CFTR is
involved, directly or indirectly, in the transport of HCO32, defects
of which could be one of the major underlying mechanisms for
CF-related clinical presentations . To investigate whether
CFTR is involved in HCO32transport in Sertoli cells, we
measured intracellular pH using a pH-sensitive fluorescent probe
BCECF-AM. The result showed that application of CFTR specific
inhibitor CFTRinh172 led to a slight acidification of the cells
(Fig. 2C). In contrast, application of forskolin, which is known to
activate CFTR, could induce alkalization of Sertoli cells, which
could be reversed by following addition of CFTRinh172 (Fig. 2C).
Simultaneous CFTRinh172 and forskolin treatment significantly
inhibited forskolin-induced alkalization (Fig. 2C). Thus, these
results suggest that CFTR may be involved in HCO32transport in
the Sertoli cells.
Involvement of CFTR-mediated HCO32transport in
FSH-stimulated cAMP production
Does the CFTR-mediated HCO32transport play any role in
spermatogenesis? Follicle-stimulating hormone (FSH) is well-
known to regulate the cAMP – CREB pathway in Sertoli cells,
which is important for spermatogenesis, through its G-protein-
coupled receptor that activates the membrane-bound adenylyl
cyclase and elevate intracellular cAMP level . Interestingly,
our previous study on sperm has demonstrated that CFTR-
mediated HCO32transport can increase intracellular cAMP by
activating the soluble adenylyl cyclase (sAC) , a proven cellular
sensor of HCO32[27,28]. Thus, the CFTR-dependent and
HCO32- activated sAC pathway might be an alternative way to
increase cAMP in Sertoli cells to regulate spermatogenesis,
provided sAC is also expressed in Sertoli cells.
Although the critical role of sAC in sperm activation has been
demonstrated [29,30], currently it is still not known whether sAC
is expressed in other cell types apart from germ cells in the testis
and whether it plays a role in spermatogenesis. RT- PCR result
showed a band with expected size of sAC in mouse and rat testis
and primary culture of rat Sertoli cells (Fig. 3A). Western blot also
exhibited a 75 kD sAC isoform expression in Sertoli cells (Fig. 3B),
which is in accordance with the splicing isoform of sAC reported
Intracellular cAMP measurement by ELISA showed that FSH
could elevate the intracellular cAMP level in the absence of
HCO32, but the presence of 25 mM HCO32could significantly
enhance the FSH-stimulated cAMP elevation (Fig. 3C). The
enhancing effect of HCO32was inhibited by both CFTRinh172
and sAC inhibitor KH7 (Fig. 3C). These results suggest that
CFTR potentiates the FSH-stimulated cAMP production, most
probably through the HCO32/sAC pathway.
Involvement of CFTR-mediated HCO32transport in FSH-
stimulated CREB phosphorylation and CREB expression
cAMP-responsive element binding protein (CREB) is a well-
known target of cAMP involved in spermatogenesis [32,33].
Coinciding with the result of cAMP assay, HCO32could also
enhance the basal and FSH-stimulated CREB phosphorylation in
the primary culture of Sertoli cells (Fig. 3D–E). The enhancing
effect of HCO32on FSH-stimulated CREB phosphorylation
could be attenuated by CFTR inhibitor (Fig. 3D–E). Similar
effect was observed in total CREB level, although without statistic
significance (Fig. 3F–G). Nevertheless, the simultaneous up-
regulation of phosphorylated and total CREB suggests that a
positive-feedback loop, similar to that in previous report, may be
present in Sertoli cells [32,33]. Interestingly, a significant
percentage of FSH-induced phosphorylation of CREB was
independent of HCO32and CFTR as shown in the HCO32free
and CFTR inhibitor-treated condition. Since FSH can also
CFTR and Spermatogenesis
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Figure 1. Defective spermatogenesis and altered expression of CREB in CF mice testis. (A) Significantly reduced testis size and weight
seen in heterozygous S489X mice compared with wild-type mice (n=3; *:p,0.05). (B) Reduced daily sperm production (DSP) values retrieved from
heterozygous S489X testes (n=4; *:p,0.05). (C) Reduced sperm numbers recovered from the epididymis of heterozygous S489X mice (+/+ n=6, +/2
n=4; *:p,0.05). (D) H&E staining of the cross sections of wild2type, heterozygous and homozygous S489X CF mice testes. Seminiferous tubules
remain intact in +/2 and 2/2 CF mice, but looser tubule contacts with larger interstitial space are observed in 2/2 testes (right) when compared
with wild type (left). Leydig cells and peritubular myloid cells are detached from most tubules in 2/2 testes. Bar =20 mm (upper panel) or 50 mm
(lower panel). (E) Statistic shows slight decrease in tubular diameter is observed in 2/2 testes. (F) Realtime PCR of protamine-2 in S489X CF mice
testes. Protamine-2 mRNA level is significantly lowered in heterozygous and homozygous S489X CF mice compared to their wildtype littermates. (G)
Immunofluorescent staining of Protamine-2 in +/+, +/2 and 2/2 S489X CF mice testes (stage VII–VIII). In +/+ testes, Protamine-2 positive signals are
located in elongated spermatids (ES) with strong immunoreactivity (red arrow). In +/2 testes, Protamine-2 is found in ES with moderate
immunoreactivity (red arrow). In 2/2 testes, there is weak intensity in the pachytene spermatocytes (P) (white arrow), ES (red arrow) and round
CFTR and Spermatogenesis
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activate Ca2+-dependent signaling pathways , the FSH-
induced HCO32/CFTR-independent CREB phosphorylation
could be contributed by the cAMP-independent intracellular
calcium signaling, which has also been demonstrated to increase
the phosphorylation of CREB . Nevertheless, bicarbonate-
mediated phosphorylation of CREB was abolished by CFTR
inhibitor treatment. These results suggest that CFTR-mediated
HCO32transport is involved in modulating the FSH-stimulated
Decreased CFTR expression and abnormal CREB
expression in human azoospermia testes
Both the in vivo and in vitro data indicate that CFTR is
involved in spermatogenesis through the activation of cAMP-
CREB signaling pathway. To confirm this pathway in human
spermatogenesis and test whether abnormality of this pathway
may contribute to pathogenesis of azoospermia,we compared the
expression profile of CFTR, CREB and the spermatogenic marker
protamine-2 in normal human and non-obstructive azoospermia
(NOA) testes. The testis sections from azoospermia patients
showed that expression of Protamine-2, a spermatid and
spermatozoa specific marker used in diagnosis of non-obstructive
(Fig. 4A&B), confirming the defect in spermatogenesis. Immu-
nofluorescence showed that CFTR was expressed in Sertoli cells
and germ cells of normal testes. However, its expression was
decreased in the testes of azoospermia patients (Fig. 4A&B).
Consistent with its down-regulation in CF mice and CFTR-
inhibited primarily cultured Sertoli cells, CBEB was also decreased
in human azoospermia testes (Fig. 4C&D), suggesting that the
CFTR-dependent CREB pathway may be important for sper-
matogenesis in human and that defect in this pathway may
represent a possible mechanism underlying the pathogenesis of
The present study has demonstrated for the first time the
involvement of CFTR in spermatogenesis and elucidated the
possible underlying signaling pathway, providing support to the
long proposed but intensively disputed link between CFTR
mutations and defects in sperm production, such as non-
obstructive azoospermia and oligospermia [13,18,19,20,37,38].
Apart from the small sample size and inconsistent clinical
observations that hampered the claims in previous studies
attempting to establish a link between CFTR mutations and
abnormality in sperm production, the main reason for the dispute
was the lack of a convincing mechanism by which CFTR is
involved in the regulation of spermatogenesis. In the present study,
we have characterized the spermatogenic phenotypes in CF mice
model, which reveals that defects of CFTR cause dysregulation of
the major regulatory pathways in spermatogenesis, such as CREB
in Sertoli cells and CREM in germ cells, resulting in impaired
sperm production. In vitro experiments further demonstrate that
CFTR can potentiate the FSH-stimulated CREB phosphorylation
by mediating HCO32transport and activation of sAC in Sertoli
cells, thus, for the first time, providing a possible mechanism for
the involvement of CFTR in spermatogenesis. Since germ cell
development critically depends on Sertoli cells, defect in CFTR-
dependent FSH-stimulated CREB activation in Sertoli cells could
affect germ cell CREM activation and germ cell development as
reflected by the diminished expression of a spermatid-specific
marker, protamine-2. It should be noted that both CREB and
CREM are known to be the master-switch transcription factors
coupled to the FSH-regulated cAMP pathway [32,39], mutations
of which are known to be related to male infertility or shown to
affect spermatogenesis [40,41]. The aberrance of both CREB and
CREM observed in CF mice testes provides a link between CFTR
mutations and defective spermatogenesis. This notion is further
supported by the observed down-regulation of CFTR and CREB
expression in human azoospermia testes, suggesting that abnormal
CFTR-CREB pathway may be a possible cause of azoospermia.
The current findings not only reveal a previously undefined role of
CFTR in regulation of spermatogenesis but also provide a possible
mechanism underlying the pathogenesis of azoospermia in non-
CF patients. Of note, there are more than 1800 mutations of
CFTR and most of them may not cause typical CF phenotypes,
but may lead to compromised spermatogenesis. Therefore, the
current findings call for more clinical studies of CFTR mutations
screening, both classic genetic mutations and other variant tracts
such as IVS8-Tn locus, in azoospermia and severe oligozoosper-
mia patients. In this regard, the rapid development of drug
targeting CFTR may provide opportunity for the possible therapy
of azoospermia caused by functional deficiency of CFTR in the
Another novel finding of the present study is the expression of
sAC in Sertoli cells and its involvement in the FSH-regulated
cAMP cascade. Although FSH is known to play an essential role in
regulating spermatogenesis through the cAMP pathway in Sertoli
cells [33,42], the present results find that full activation of the
FSH-stimulated cAMP pathway depends on HCO32, sAC and
CFTR, suggesting that the CFTR-HCO32-dependent cAMP
pathway is not merely an alternative or redundant pathway. In
fact, the CFTR-dependent sAC-activated cAMP pathway may be
an important loop in the FSH-regulated cAMP cascade for
spermatogenesis since mutation or abnormal expression of CFTR
could result in impaired spermatogenesis or azoospermia. An
increasing body of evidence obtained from other somatic cells,
such as lung epithelial cell line Calu-3, indicates that sAC may also
be expressed in particular cell microdomain and nucleus as a more
fine-tuned and sensitive way to regulate cAMP production and the
down-stream target, CREB . The activation of membrane-
bound AC by G protein-coupled receptors, such as FSH receptor,
may in turn activate CFTR, a cAMP-activated anion channel,
allowing for entry of HCO32and subsequent activation of sAC in
other cellular compartments. This may represent a more rapid and
economic way for cells to response to extracellular or intracellular
stimuli , such as FSH and testosterone during spermatogen-
The present finding that inhibition of CFTR leads to down-
regulated FSH-activated cAMP-CREB signaling pathway has also
shed new light on the pathogenesis of other CF-related diseases
spermatids (RS) (yellow arrow). Bar =50 mm. (H) Immunofluorescent staining of CREM in S489X CF mice testes (stage VIII–I). In wild-type testes, CREM
positive signals are localized in RS (white arrow), with strong immunoreactivity. In +/2 and 2/2testis, CREM showed weak immunoreactivity. Bar
=50 mm. (I) Immunofluorescent staining of CREB in S489X CF mice testes shows stronger immunoactivity in +/+ and +/2 mice Sertoli cell compared
to that of 2/2. Bar =50 mm. (J) Western blot analysis of CREM expression in S489X CF testes. CREM expression is decreased in +/2 and 2/2 testes
compared to wild-type (n=3; *:p,0.05). (K) Western blot analysis of CREB expression in S489X CF testes. CREB expression is decreased in 2/2 testes
compared to +/2 and wild-type (+/+ n=2, +/2 and 2/2 n=3; **:p,0.01). Statistic data are shown as mean6SEM.
CFTR and Spermatogenesis
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CFTR and Spermatogenesis
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beyond the reproductive tract, since CREB is a master
transcription factor capable of regulating a variety of genes
involved in different cellular events, including metabolism,
neurotransmission, cell cycle/cell survival, cell growth, signal
transduction and transport . Interestingly, a recent study has
shown dampened beta-adrenergic receptor-activated cAMP path-
ways in CF cells . The dampened CFTR-dependent cAMP-
CREB signaling pathway in response to hormone stimuli, such as
adrenalin and FSH, may lead to impaired hormonal regulation
and downstream physiological processes, such as epithelial ion
transport and spermatogenesis. This may be responsible, at least in
part, for the pathophysiological alterations seen in CF. Consider-
ing the broad spectrum of CREB target genes and their
involvement in a wide range of physiological processes , the
defective CFTR-dependent CREB pathway and the resulting
abnormal expression of its downstream targets may represent an
important mechanism underlying various CF-related diseases, and
thus, a major therapeutic target for the diseases, including male
Materials and Methods
Animal and cell culture
cftrtm1Unc(S489X) mice  were from Jackson’s laboratory and
maintained in LASEC of CUHK. 2–6 months old mutant mice
and their wild type littermate as control groups were used.
For the primary Sertoli cell culture, 20-day old male S-D rats
were used for Sertoli cells isolation as described previously .
Hypotonic treatment was performed 2 days after isolation to lyse
residual germ cells, and obtained a purity .95%. All the animals
handling protocol was approved by the Animal Research Ethics
Committee of the University (Ref. No: 04/025/ERG; CUHK
Human testicular tissue samples
Biopsy materials were taken from patients with a normal
karyotype who attended the ART (Assisted Reproduction
Techniques) Clinic at Peking University Shenzhen Hospital. Prior
to any data collection, the experimental protocol was reviewed and
approved by the ethics committee of the hospital (Ref. No:
20090018) and all patients signed informed consent approving the
use of their tissues for research purposes.
A total of 11 men aged between 23 and 45 years were
included in the study. Samples were collected by Department of
included had no health problems other than azoospermia.
Testicular specimens were obtained from the patients with non-
obstructive azoospermia with the open micro-testicular biopsy
technique. Informed consents were obtained from all patients
prior to participation in the study. The original reports of the
pathologists were reviewed. Johnsen’s score count was used for
histological characterization. Normospermia samples (Johnsen’s
score=8) and azoospermia samples (Johnsen’s score#4) were
used. The specimens (n=6) were obtained from the patients
orchiectomized for the following diagnoses: cysta dermoides,
cystadenoma, and prostate cancer.
Monoclonal anti-CFTR MAb (CF3) was purchased from Enzo
Life Sciences (Farmingdale, NY). Monoclonal rabbit anti-CREB,
polyclonal rabbit anti-Phospho-CREB (Ser133) was purchased
from Cell Signaling Technology (Beverly, MA). polyclonal goat
anti-CREM (C-12), polyclonal goat anti-Protamine 2 (C-14) was
purchased from Santa Cruz biotechnology (Santa Cruz, CA).
Polyclonal rabbit anti-sAC antibody was a gift from Dr. Ping Bo
Clinical tissue specimens and CF mice testis specimens were
fixed by formalin, embedded in paraffin and cut into 3-mm
sections. Paraffin sections were dewaxed in xylene and rehydrated
in descending concentrations of alcohol. Antigen retrieval was
achieved by incubation in sub-boil citrate buffer (pH 6.0). All
slides were incubated with 3% H2O2for 10–15 minutes to block
the endogenous peroxidase. After slides were washed with PBS,
nonspecific background staining was blocked by 5% normal goat
serum (Santa Cruz) for 30 min, followed by overnight incubation
at 4uC with anti-CREB (1:200). For negative control, primary
antibodies were omitted. UltraVision ONE Detection System
HRP Polymer & DAB Plus Chromogen (Thermo Fisher
Scientific Inc.) was used for detection according to the
manufacturer’s instructions. Mayer’s hematoxylin was used for
In quantitative analysis of CREB expression in azoospermia
sample, the percentage of CREB-positive Sertoli cells in ten
random fields were compared for each sample. Data from 5
normal human and 6 azoospermia patients were used for statistic.
Paraffin sections including clinical tissue specimens and CF
mice testis specimens were treated as above. Then the sections
were incubated with appropriate diluted primary antibody (anti-
CFTR 1:200; anti-CREM 1:200; anti-Protamine-2 1:200; anti-
CREB 1:200) at 4uC overnight, washed with PBS, then incubated
with secondary antibody (Alexa 568-conjugated goat-anti-mouse
IgG for CFTR, Alexa 488-conjugated rabbit-anti-goat IgG for
CREM and Protamine-2, Alexa 488-conjugated goat-anti-rabbit
for CREB, from Molecular Probes, Eugene, OR) at 1:500 dilution
in PBS for 1 hour at room temperature. After washed with PBS,
the sections were counterstained with Hoechst 33258 for 20 min
and covered with ProlongH Gold Antifade Reagent (Invitrogen,
Quantitative analysis of human azoospermia samples was
performed by Metamorph software, with average optical density
of ten random fields were compared for each sample. Data from 5
normal human and 6 azoospermia patients were used for statistic.
Protein was extracted by RIPA cell lysis buffer. 40,100 mg
proteins were resolved by SDS-PAGE followed by transferring
onto nitrocellulose membranes. Membranes were blocked with
4% milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 hour,
and then incubated in primary antibodies (anti-CFTR 1:500, anti
Figure 2. Functional expression of CFTR in rat Sertoli cell primary culture. (A) RT-PCR shows CFTR mRNA expression in rat Sertoli cells
(expected PCR product size: 481 bp). (B) Western blot detects a band at around 160 kDa, showing CFTR protein expression in rat Sertoli cells. (C)
Examination of CFTR-involved HCO32transport by intracellular pH (pHi) measurement. 10 mM CFTRinh172 results in a slight acidification of pHi at
basal condition. 10 mM forskolin induces a rapid alkalization in pHi, which can be reversed by following addition of CFTRinh172. Simultaneous
forskolin and CFTRinh172 treatment significantly inhibits forskolin-induced alkalization. (DMSO n=4, CFTRinh172 n=3, Forskolin n=8,
CFTRinh172+Forskolin n=7; *: p,0.05, **:p,0.01,***: p,0.001, compared to Forskolin). Statistic data are shown as mean6SEM.
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sAC 1:500, anti-CREB 1:200, anti-phospho-CREB 1:200, anti-
CREM 1:200) in 2% milk at 4uC overnight. Membranes are
washed three times in TBS-T followed by incubation with anti-
mouse IgG-HRP (1:10000), anti-rabbit IgG-HRP (1:10000) or
donkey anti-goat IgG-HRP (1:5000) in 2% milk for 1 hour at
room temperature. Following three washes in TBS-T, proteins are
detected using an enhanced chemiluminescence kit according to
the manufacturer’s instructions.
Figure 3. Involvement of CFTR-mediated HCO32transport in FSH-stimulated cAMP production and CREB regulation. (A) RT-PCR
result shows the expression of sAC in mouse testis (mT), rat testis (rT) and primary Sertoli cells (SC) (expected PCR product size: 119 bp). (B) Western
blot shows the expression of a 75 kD isoform of sAC in the Sertoli cells (SC). Adult rat germ cell (GC) sample shows predominatly exression of 50 kD,
with another unidentified band. (C) Effect of HCO32, CFTR and sAC inhibitors on 50 ng FSH-stimulated cAMP production, with presence of 25 mM
HCO32potentiating FSH-stimulated cAMP production in Sertoli cells, which is abolished by 10 mM CFTRinh172 and 10 mM sAC inhibitor KH7. (n=3;
***:p,0.01). (D-E) Effect of HCO32and CFTR inhibitors on FSH-stimulated CREB phosphorylation. Western blot results shows that 25 mM HCO32
potentiates 50 ng FSH-stimulated CREB phosphorylation in Sertoli cells, while 10 mM CFTRinh172 attenuates the effect of HCO32(n=3; *:p,0.05;
***:p,0.001). (F-G) Effect of HCO32and CFTR inhibitor on CREB expression. Western blot results shows that removal of HCO32or treatment with
CFTRinh172 slightly decrease total CREB level in the presence of FSH, but without statistic significance (n=3). Statistic data are shown as mean6SEM.
Detailed protocol in Materials and Methods.
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CFTR and Spermatogenesis
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Intracellular pH measurement
Intracellular pH was measured by BCECF fluorescent imaging.
1 mM BCECF-AM (Molecular Probe) was loaded to cells in
Krebs–Henseleit (K-H) solution (in mM, 117 NaCl, 4.7 KCl, 2.56
CaCl2, 1.2 MgSO4, 24.8 NaHCO3, 1.2 KH2PO4, and 11.1
glucose, pH 7.4) at 37uC for 20 min. During measurement, the
cells were incubated in Cl2- free K-H solution (in mM, 117 Na-
gluconate, 4.7 K-gluconate, 1.2 MgSO4, 1.2 KH2PO4, 24.8
NaHCO3, 20 Ca-gluconate, and 11.1 glucose, pH 7.4, gassed with
5% CO2and kept at 37uC). The fluorescent dye was alternately
excited with two wavelengths (440 nm and 490 nm) and the
emission was measured at 510 nm. The ratio of two signals
(440 nm:490 nm) is directly proportional to pH.
Sertoli cells grown in 24-well plate were deprived from HCO32
by treatment with HCO32-free DMEM/F12 medium for 2 hr.
Then the medium was change to HCO32-free or 25 mM HCO32
DMEM/F12 and incubated for 15 min. In some experiment
groups, 10 mM CFTRinh172 (Sigma), 10 mM KH7 (ChemDiv),
50 ng recombinant FSH were added to the media. 100 mM IBMX
(Sigma) was supplemented to all media to inhibit the cAMP
degradation. After treatment, the cells were lyzed with 0.1 M HCl
and centrifuged at 1000 g for 10 min to remove the cell debris.
cAMP assay was performed with the cell lysate by enzyme
immunoassay following the EIA kit (Assay Design, 901–066)
CREB phosphorylation assay
Sertoli cells were deprived from HCO32by treatment with
HCO32-free DMEM/F12 medium for 2 hr. Then the medium
was change to HCO32-free or 25 mM HCO32DMEM/F12 and
incubated for 30 min. In some experiment groups, 10 mM
CFTRinh172 (Sigma) or/and 50 ng recombinant FSH were
added to the media. Whole cell protein was extracted by RIPA
buffer with protease inhibitor cocktail and sodium orthovanadate.
CREB and phospho-CREB was analyzed by Western blot.
For two groups of data, two-tail unpaired Student’s t tests were
used. For three or more groups, data were analyzed by one-way
ANOVA and Tukey’s post hoc test. A probability of P ,0.05 was
considered to be statistically significant.
spermatids of CF mice. Decreased size of spermatocytes and
round spermatids in stage V–VI is observed in 2/2 testis
compared to +/+ and +/2. The cytoplasmic area of spermatocytes
and round spermatids shrink progressively from +/2 to 2/2
testes. P: pachytene spermatocytes, RS: round spermatids.
Enlarged image of spermatocytes and round
Conceived and designed the experiments: WMX HCC. Performed the
experiments: WMX JC HC RYD JDD TTS WYC MKY XHZ LLT KLF.
Analyzed the data: WMX JC HC KLF HCC. Contributed reagents/
materials/analysis tools: AL QXS QHS PBH. Wrote the paper: HCC
1. Anderson MP, Gregory RJ, Thompson S, Souza DW, Paul S, et al. (1991)
Demonstration that CFTR is a chloride channel by alteration of its anion
selectivity. Science 253: 202–205.
2. Poulsen JH, Fischer H, Illek B, Machen TE (1994) Bicarbonate conductance and
pH regulatory capability of cystic fibrosis transmembrane conductance
regulator. Proc Natl Acad Sci U S A 91: 5340–5344.
3. Rowe SM, Miller S, Sorscher EJ (2005) Cystic fibrosis. N Engl J Med 352:
4. Quinton PM (1990) Cystic fibrosis: a disease in electrolyte transport. FASEB J 4:
5. Chan HC, Ruan YC, He Q, Chen MH, Chen H, et al. (2009) The cystic fibrosis
transmembrane conductance regulator in reproductive health and disease.
J Physiol 587: 2187–2195.
6. Chillon M, Casals T, Mercier B, Bassas L, Lissens W, et al. (1995) Mutations in
the cystic fibrosis gene in patients with congenital absence of the vas deferens.
N Engl J Med 332: 1475–1480.
7. Tizzano EF, Silver MM, Chitayat D, Benichou JC, Buchwald M (1994)
Differential cellular expression of cystic fibrosis transmembrane regulator in
human reproductive tissues. Clues for the infertility in patients with cystic fibrosis
Am J Pathol 144: 906–914.
8. Hihnala S, Kujala M, Toppari J, Kere J, Holmberg C, et al. (2006) Expression of
SLC26A3, CFTR and NHE3 in the human male reproductive tract: role in male
subfertility caused by congenital chloride diarrhoea. Mol Hum Reprod 12: 107–111.
9. Gong XD, Li JC, Cheung KH, Leung GP, Chew SB, etal. (2001) Expression of the
cystic fibrosis transmembrane conductance regulator in rat spermatids: implication
for the site of action of antispermatogenic agents. Mol Hum Reprod 7: 705–713.
10. Boockfor FR, Morris RA, DeSimone DC, Hunt DM, Walsh KB (1998) Sertoli
cell expression of the cystic fibrosis transmembrane conductance regulator.
Am J Physiol 274: C922–930.
11. Trezise AE, Linder CC, Grieger D, Thompson EW, Meunier H, et al. (1993)
CFTR expression is regulated during both the cycle of the seminiferous
epithelium and the oestrous cycle of rodents. Nat Genet 3: 157–164.
12. Larriba S, Bassas L, Gimenez J, Ramos MD, Segura A, et al. (1998) Testicular
CFTR splice variants in patients with congenital absence of the vas deferens.
Hum Mol Genet 7: 1739–1743.
13. van der Ven K, Messer L, van der Ven H, Jeyendran RS, Ober C (1996) Cystic
fibrosis mutation screening in healthy men with reduced sperm quality. Hum
Reprod 11: 513–517.
14. Jakubiczka S, Bettecken T, Stumm M, Nickel I, Musebeck J, et al. (1999)
Frequency of CFTR gene mutations in males participating in an ICSI
programme. Hum Reprod 14: 1833–1834.
15. Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, et al.
(2002) Genetic risk factors in infertile men with severe oligozoospermia and
azoospermia. Hum Reprod 17: 13–16.
16. Tuerlings JH, Mol B, Kremer JA, Looman M, Meuleman EJ, et al. (1998)
Mutation frequency of cystic fibrosis transmembrane regulator is not increased
Figure 4. Reduced CFTR and CREB expression in human azoospermia testes. (A) Representative immunoinfluoresence staining of CFTR
(red) and Protamine-2 (green) in normal human (n=5) and azoospermia patients (n=6) testes, counterstained with Hoechst (blue). In the normal
human testes, Protamine-2 positive signals are localized in the round spermatids (RS, yellow arow) and elongated spermatids (ES, white arrow)
(Stages I–II); CFTR positive signals are localized in pachytene spermatocytes (P, blue arrow), RS and Sertoli cells (SC, green arrow) (stages I–VI),
especially in RS. In azoospermia testes, no positive signal of Protamine-2 and weak signals of CFTR are detected (Magnification: 400x). Bar =50 mm.
(B) Statistic analysis of protamine-2 and CFTR positive signal area in normal (n=5) and azoospermia (n=6) human testes samples (***:p,0.001). (C)
Immunohistochemistry of CREB in normal human and azoospermia testes. In normal human testes, CREB positive signals are localized in RS (red
arrow) and SC (blue arrow). CREB inmmunoreativity in SC is decreased in the testes with spermatogenic arrest obtained from azoospermia patients.
Bar =50 mm. (D) Statistic analysis of CREB positive Sertoli cell percentage in normal (n=5) and azoospermia (n=6) human testes (***:p,0.001).
Statistic data are shown as mean6SEM.
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in oligozoospermic male candidates for intracytoplasmic sperm injection. Fertil
Steril 69: 899–903.
17. Pallares-Ruiz N, Carles S, Des Georges M, Guittard C, Arnal F, et al. (1999)
Complete mutational screening of the cystic fibrosis transmembrane conduc-
tance regulator gene: cystic fibrosis mutations are not involved in healthy men
with reduced sperm quality. Hum Reprod 14: 3035–3040.
18. Mak V, Zielenski J, Tsui LC, Durie P, Zini A, et al. (2000) Cystic fibrosis gene
mutations and infertile men with primary testicular failure. Hum Reprod 15:
19. Ravnik-Glavac M, Svetina N, Zorn B, Peterlin B, Glavac D (2001) Involvement
of CFTR gene alterations in obstructive and nonobstructive infertility in men.
Genet Test 5: 243–247.
20. Kiesewetter S, Macek M, Jr., Davis C, Curristin SM, Chu CS, et al. (1993) A
mutation in CFTR produces different phenotypes depending on chromosomal
background. Nat Genet 5: 274–278.
21. Ashby J, Tinwell H, Lefevre PA, Joiner R, Haseman J (2003) The effect on
sperm production in adult Sprague-Dawley rats exposed by gavage to bisphenol
A between postnatal days 91-97. Toxicol Sci 74: 129–138.
22. Hess RA, de Franca LR (2008) Spermatogenesis and Cycle of the Seminiferous
Epithelium.: In C.Y. C, ed. Molecular Mechanisms in Spermatogenesis. New
Yorl: Landes Bioscience. pp 1–15.
23. Mruk DD, Cheng CY (1999) Sertolin is a novel gene marker of cell-cell
interactions in the rat testis. J Biol Chem 274: 27056–27068.
24. Quinton PM (2001) The neglected ion: HCO3. Nat Med 7: 292–293.
25. Tindall DJ, Tash JS, Means AR (1981) Factors affecting Sertoli cell function in
the testis. Environ Health Perspect 38: 5–10.
26. Xu WM, Shi QX, Chen WY, Zhou CX, Ni Y, et al. (2007) Cystic fibrosis
transmembrane conductance regulator is vital to sperm fertilizing capacity and
male fertility. [see comment] Proceedings of the National Academy of Sciences
of the United States of America 104: 9816–9821.
27. Xie F, Conti M (2004) Expression of the soluble adenylyl cyclase during rat
spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ
cells. Dev Biol 265: 196–206.
28. Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, et al. (2000) Soluble
adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:
29. Esposito G, Jaiswal BS, Xie F, Krajnc-Franken MA, Robben TJ, et al. (2004)
Mice deficient for soluble adenylyl cyclase are infertile because of a severe
sperm-motility defect. Proc Natl Acad Sci U S A 101: 2993–2998.
30. Marquez B, Suarez SS (2008) Soluble adenylyl cyclase is required for activation
of sperm but does not have a direct effect on hyperactivation. Reprod Fertil Dev
31. Geng W, Wang Z, Zhang J, Reed BY, Pak CY, et al. (2005) Cloning and
characterization of the human soluble adenylyl cyclase. Am J Physiol Cell
Physiol 288: C1305–1316.
32. Don J, Stelzer G (2002) The expanding family of CREB/CREM transcription
factors that are involved with spermatogenesis. Mol Cell Endocrinol 187:
33. Walker WH, Habener JF (1996) Role of transcription factors CREB and CREM
in cAMP-regulated transcription during spermatogenesis. Trends Endocrinol
Metab 7: 133–138.
34. Walker WH, Cheng J (2005) FSH and testosterone signaling in Sertoli cells.
Reproduction 130: 15–28.
35. Mayr B, Montminy M (2001) Transcriptional regulation by the phosphoryla-
tion-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599–609.
36. Song GJ, Lee H, Park Y, Lee HJ, Lee YS, et al. (2000) Expression pattern of
germ cell-specific genes in the testis of patients with nonobstructive azoospermia:
usefulness as a molecular marker to predict the presence of testicular sperm.
Fertil Steril 73: 1104–1108.
37. Larriba S, Bonache S, Sarquella J, Ramos MD, Gimenez J, et al. (2005)
Molecular evaluation of CFTR sequence variants in male infertility of testicular
origin. Int J Androl 28: 284–290.
38. Schulz S, Jakubiczka S, Kropf S, Nickel I, Muschke P, et al. (2006) Increased
frequency of cystic fibrosis transmembrane conductance regulator gene
mutations in infertile males. Fertil Steril 85: 135–138.
39. De Cesare D, Fimia GM, Sassone-Corsi P (2000) CREM, a master-switch of the
transcriptional cascade in male germ cells. J Endocrinol Invest 23: 592–596.
40. Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M, et al. (1996)
Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice.
Nature 380: 159–162.
41. Scobey M, Bertera S, Somers J, Watkins S, Zeleznik A, et al. (2001) Delivery of a
cyclic adenosine 3’,5’-monophosphate response element-binding protein (creb)
mutant to seminiferous tubules results in impaired spermatogenesis. Endocri-
nology 142: 948–954.
42. Dorrington JH, Roller NF, Fritz IB (1975) Effects of follicle-stimulating hormone
on cultures of Sertoli cell preparations. Mol Cell Endocrinol 3: 57–70.
43. Bundey RA, Insel PA (2004) Discrete intracellular signaling domains of soluble
adenylyl cyclase: camps of cAMP? Sci STKE 2004: pe19.
44. Mak JC, Chuang TT, Harris CA, Barnes PJ (2002) Increased expression of G
protein-coupled receptor kinases in cystic fibrosis lung. Eur J Pharmacol 436:
45. Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, et al.
(1992) An animal model for cystic fibrosis made by gene targeting. Science 257:
CFTR and Spermatogenesis
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