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Interference
with the formation of the epididymalmicroenvironmenta new strategy for
male contraception?
X.D.
Gong, G.P.H. Leung, B.L.Y. Cheuk,
P.Y.D. Wong
Department
of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong
Kong
Asian J Androl 2000 Mar; 2: 39-45
Keywords:
epididymis;
cyclooxygenase; contraception; cystic fibrosis transmembrane conductance
regulator
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Recent work in our laboratory has shown that this protein is also expressed by developing germ cells indicating a role of this protein in spermatogenesis. In view of the fact that the CFTR gene has a far reaching and widespread effeon human reproduction, understanding the role of CFTR in the male reproductive tissues and its intervention by pharmacological agents can open a new avenue of research into the development of novel male contraceptives.1 Introduction
It was thirty years ago when Orgebin-Crist[1] discovered that spermatozoa have to go through maturational changes in the epididymis before they can fertilize the ovum. Since then a lot of attention have been focussed on the physiology and biochemistry of the epididymis. This sudden blitz of effort on epididymal research was largely driven by the recognition that contraceptive agents which interfere with sperm maturation in the epididymis have many advantages over those which suppress sperm production in the testis. The former agents are expected to have a quick onset of action, rapid reversibility upon withdrawal, and reduced chances of mutagenic damage and endocrine impairment of libido. These advantages have been borne by -chlorohydrin[2] and the chlorinated sugars[3] which act by interefering with sperm metabolism in the epididymis. However, interest in these agents as a potential male contraceptive soon waned as these compounds were found to be neurotoxic (although recently there is a revival of interest based on synthesis of analogues lacking neurotoxicity). In recent years, the advent of molecular biology techniques have led to the identification and cloning of genes encoding sperm-coating or epididymal-specific proteins which can be targeted for immunocontraception. However, for these methods to be effective, organ-specific delivery methods of antisense oligonucleotides/antibodies will have to be developed. Reduction of the prominent consitituents of the epididymal fluid viz. -glucosidase and L-carnitine or enhancement of sperm transport through the epididymis by pharmacological means has not led to infertility in animals[4]. Despite the consensus held that the epididymis is indispensible for the full expression of male fertility, attempts to induce infertility via an epididymal approach remain elusive. It is clear that new initiatives are necessary to generate new leads for effective contraception in the male.2 Formation of the epididymal microenvironment
The epididymis plays an indispensible role in male reproduction by forming an optimal fluid environment in which spermatozoa undergo maturational changes before they acquire their full fertilizing capacity. It has been established in our laboratory that the epithelial cells lining the epididymis secrete chloride and bicarbonate when stimulated by physiological neurohumoral stimuli[5-9]. This active transport of anions creates the driving force for cation secretion, and secondarily water secretion down an osmotic gradient. The movement of electrolytes and water across the epithelium determines the fluidity of the luminal environment in which spermatozoa are bathed. A correct fluid environment is essential to the normal development and full expression of sperm fertilizing capacity.3 Control of electrolyte secretion through cyclooxygenase (COX)
The
formation of the epididymal fluid environment is regulated by
nerves and hormones which act in a timely and concerted manner to control
electrolyte and water transport across the epididymal epithelium. A common
feature shared by many of these
physiological regulators is that their effects can be blocked by the non-steroidal
anti-inflammatory drugs (NSAIDs), which are inhibitors of prostaglandin
synthesis, suggesting that prostaglandins are the common mediators of
the responses[9-13]. Recently, we have proposed a model to
explain the stimulation of anion
secretion by bradykinins and other peptide hormones in the rat epididymis[9](Figure
1). According to this model, these
peptides act on receptors on the basal
cells to activate PLA2 with release of arachidonic acid (AA)
from membrane phospholipids. AA is converted to
PGG2 and PGH2 by the cyclo-oxygenase 1 (COX-1)
and then to PGE2 by the specific isomerase.
PGE2 diffuses out of the cells and acts on the prostaglandin
receptors, notably the EP2/4 subtypes, on the basolateral membrane of
the principal cells to increase intracellular cAMP which activates an
apical anion channel (CFTR) resulting in secretion of anions and, secondarily,
water. This model is based on the
evidence that (1) COX-1 specific inhibitors inhibit the LBK-stimulated
anion secretion. (2) PGE2, and not the other COX products mimicked
the effect of LBK. (3) The effect of PGE2 is mediated through
the cAMP-coupled EP2/4 receptors as its effect is reproduced by 11-deoxyl
PGE1, a specific EP2/4 receptor agonist, but not by sulprostone,
a specific EP1/3 receptor agonist. (4) COX-1 mRNA is expressed by the
intact rat epididymis and the COX-1 protein is localized mainly in the
basal cells. (5) bradykinin caused PGE2
release from epididymal epithelia and along with the bradykinin-induced
anion secretion is blockable by COX-1 selective inhibitors[14].
Through forming PGE2, cyclooxygenase (COX-1) regulates
anion and fluid secretion and
optimize the milieu in which sperm are bathed[15]. The corollary
to these findings is that
inhibiton of the COX-1 isozyme by the non-steroidal anti-inflammatory
drugs (NSAIDs) should disrupt
the epididymal environment and impair fertility.
4 Effects of NSAIDs on epididymal sperm motility and spermatocrit
The
role of cyclooxygenase in the fomation of the epididymal fluid was assessed
by treating male rats with NSAIDs and their fertility evaluated by mating
study. It is well
known that prostanoids have a physiological role in protecting the gastric
mucosa from haemorrhage and ulceration.
Inhibition of the enzyme by
COX-1 inhibitors would remove the protective action and result
in gastric damage[16].
We treated rats with indomethacin, a COX-1 specific inhibitor,
at different dose
regimens and found
no effect on fertility (unpublished), percentage motile sperm and spermatocrit
(a measure of fluid content in the epididymis[17-19])(Table
1), even at a dose rate of 30 mg/kg/day for 3 consecutive days (this dose regimen
was found to cause substantial gastric mucosal damage).
Negative results were also obtained with 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone
(DFU), a COX-2 specific inhibitor[20](Table 1). This lack of
effect of NSAIDs is explicable in light of the alternative pathways which
can drive secretion when the COX pathway is suppressed. It is of interest
to note that mice
in which COX-1 has been made deficient by gene knockout were found to
be fertile[21].
Table
1. Effects of COX inhibitors.
|
Drug |
Dose
regimen |
Spermatocrit |
%
motile sperm |
|
Control |
- |
62%2.4% |
70%4.5% |
|
Indomethacin |
3mg/kg/day,
3 days |
56%1.3% |
65%3.8% |
|
3mg/kg/day,
10 days |
60%2.6% |
66%3% |
|
|
3mg/kg/day,
30 days |
66%3.5% |
68%3.5% |
|
|
10mg/kg/day,
10 days* |
58%5.5% |
62%1.5% |
|
|
30mg/kg/day,
3 days* |
65%4.5% |
68%2.5% |
|
|
DFU** |
3mg/kg/day,
10 days |
63%2.5% |
65%1.4% |
|
10mg/kg/day,
10 days |
60%2.9% |
65%1.3% |
|
|
10mg/kg/day,
30 days |
61%1.3% |
63%2.3% |
*Rats
showing gastric damage at these dose levels.
**5, 5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone.
Spermatocrit was measured by method previously described[17-19]
and sperm motility
was measured by TOX Integrated Visual Optical System for Sperm Analysis
(Hamilton Thorne Research, MA 01915, USA).
5
Other COX-independent pathways
As
with many physiological processes, there are multiple
regulating pathways controlling secretion of electrolytes and fluid
in the epididymis. Blocking one of them
will not lead to severe arrest of secretion as the other pathways compensate
and make up for the loss. Other COX-independent pathways also exist in
the epididymis[8,14,22]. Several hormones and neurotransmitters
can stimulate secretion by directly raising cAMP through receptorG proteincoupled
adenylate cyclase. Furthermore, a number of secretory agonits stimulate
secretion by raising intracellular calcium[7,14,23-30].
Although many control pathways differ in their G-protein-coupled
events, they all
terminate on the apical
membrane of the epididymal epithelial cells where they increase
the conductance of the apical membrane to anions (chloride and bicarbonate)
through activation of chloride channels.
Several anion channels with different characteristics have been
described[31-33] but the one which has great relevance to transepithelial
secretion of electrolytes and fluid is a small conductance cAMP-activated
chloride channel, also known as the cystic fibrosis transmembrane conductance
regulator (CFTR)[34-37]. This
channel plays an integral role
in the secretion of electrolytes and fluid
and hence determines the fluidity of the microenvironment in which
maturing spermatozoa are bathed.
6
The cystic fibrosis transmembrane conductance regulator (the cAMP-activated
chloride channels)
It
is known that an anion channel encoded by the cystic fibrosis transmembrane
conductance regulator (CFTR) gene plays a pivotal role in anion secretion
hence the formation of epididymal fluid environment[38].
The evidence for the importance of this membrane protein has been
borne by the genetic disease cystic fibrosis (CF).
In CF, mutation
of the CFTR gene has led to abnormal luminal environment associated with
blockage or agenesis of the epididymis and vas. CF is a very heterogenous
genetic disease with about 700 mutations known. These myrids of mutations
have given rise to a wide range of phenotypes with varied degree
of disease severity.
For example, the most severe forms of mutation (508)
(thought to be associated
with the most severe loss of CFTR function) have led to clinical CF.
Men carrying these mutations suffer from debilitating respiratory
and pancreatic insufficiency, and also infertility.
In these patients, respiratory failure accounts for the morbidity
and mortality of the disease. The milder forms of mutation (thought to
be associated with moderate loss of CFTR function), on the other hand,
are associated with congenital bilateral absence of the vas deferens (CBVAD)
as the only disease manifestation[39-44]; men carrying these
mutations have apparently normal lung and pancreatic functions.
Furthermore, in
the least severe forms of the disease (associated with minimal loss of
CFTR), poor sperm quality appears to be the only sign in apparently normal
healthy men[45] (Figure
2). These observations
may imply that the male reproductive system is highly dependent
on CFTR for its normal function and consequently most vulnerable to modest
loss of CFTR. Cystic fibrosis
exemplifies the importance of a membrane transport protein in reproduction
and demonstrates the principle that disruption of this protein can compromise
fertility.
Figure
2. Cystic fibrosis transmembrane conductance regulator (CFTR) mutations
are responsible for poor sperm quality in healthy males with cystic fibrosis
and congenital bilateral absence of the vas deferens (CBAVD). The CFTR
gene has a far-reaching effect on human reproduction.
7
Pharmacological intervention of CFTR in epididymis
Given the importance of CFTR in the formation of the epididymal fluid, pharmacological intervention of CFTR activity could, in principle, lead to alterations of the sperm microenvironment. As with other ion channels on cell membrane, CFTR is amenable to regulation by pharmacological agents[46]. Recently, we reported that genistein, a flavonoid, increases chloride and bicarbonate secretion in the epididymis when added to the apical membrane of the epididymal epithelia. The genistein-induced secretion was observed in basolaterally permeabilized epithelia (Figure 3) and blocked by non-specific chloride channel blockers, diphenylamine-2-carboxylate (DPC), but not by the Ca2+ -activated Cl- channel blocker, 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS), suggesting that the secretion response was mediated by CFTR. Unlike other physiological agonists, genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the the action of genistein. Furthermore, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the secretory response to genistein, but the response was restored upon addition of a modest amount (not sufficient to elicit a significant response by itself) of exogenous cAMP. These results suggest the action of genistein is complex and may require CFTR to be in a phosphorylated form before it can exert its stimulating effect. Arising from these experimental results is the proposition that genistein may provide therapeutic benefit to male infertility associated with CF[47].
Figure 3. Effect of MDL-12330A (adenylate cyclase inhibitor) on short-circuit current (Isc) (chloride secretion) response to genistein. Tissue was first treated with nystatin (500 M) added basolaterally for 45 min to permeabilize the basolateral membrane, then with MDL-12330A (50 mM) added basolaterally. Genistein (20 M) was then added apically with (C) or without (B) replenishment with cAMP (5 M). Control response to genistein in basolaterally permeabilized epithelium is shown in (A). Horizontal lines indicate zero Isc[47].
In
a contrary manner, blockers of CFTR could be used to disrupt epididymal
microenvironment by blocking fluid secretion.
There are putative blockers of CFTR at present,
but they are neither selective nor potent[46,48].
We have explored some new blockers of epididymal CFTR
with a view to developing
them into potential antifertility agents for men. We found the antifertility
agents lonidamine and its analogue AF 2785 are potent blockers (more potent
than the putative chloride channel blockers)
of CFTR in the epididymis[49]. The inhibition by lonidamine
and AF2785 of the channel should lead to inhibition of transepithelial
secretion of chloride (and secondarily fluid).
Cultured rat epididymal epithelia have been used as a model to
study transepithelial secretion of electrolytes in the epididymis[50,51].
When stimulated with exogenous cAMP or cAMP-elevating agonists, these
epithelia respond by an increase in electrogenic chloride secretion (measured
as short-circuit current, Isc)[50,52] which can be blocked
by putative chloride
channel blockers, such as the arylaminobenzoates and sulfonylureas[48].
Apically applied lonidamine and AF2785 were found to inhibit the cAMP-induced
short-circuit current[49], in keeping with their effects on
chloride conductance. As with the whole-cell patch clamp study, they were
more potent than the conventional chloride channel blockers as evaluated
by the IC50 values. These results are promising as they point
to the potential use of these compounds in blocking fluid secretion by
the epididymis. By virtue of their inhibition of the formation of the
epididymal microenvironment, they can evolve into novel male contraceptives.
Systematic screening of indazole compounds based on lonidamine and AF2785
for their ability to block epididymal CFTR will prove fruitful.
8
CFTR in germ cells
In
addition to anomalies in the epididymis and vas, men with CF are known
to have reduced germ cell number in the testis and many cell types are
malformed[53,54]. This may infer the seminiferous epithelium
being the other infliction site
of the disease. Evidence
are available which suggest
the Sertoli cells from the
rat testes, like the epididymal cells, secrete anions upon stimulation
with cAMP[55]. In line with this observation is the finding
that Sertoli cells express
CFTR[56]. Buchwald
and his colleagues[57,58] using in situ hybridization have
shown CFTR mRNA associated with developing germ cells in the rat testis
but there has been no functional study to demonstrate the presence of
CFTR in germ cells.
Using
whole-cell patch clamp technique we have identified a cAMP-activated chloride
conductance in germ cells isolated from the rat testes (Figure
3). Although the
exact spermatogenic stages of the germ cells exhibiting the CFTR current
was not known with certainty at present, they appeared to be at
the round spermatid stage.
Preliminary study revealed the cAMP-activated current has a linear
voltage/current relationship (Figure
4) and sensitivity to putative
chloride channel blocker (unpublished), consistent with it being a
cAMP-activated chloride current. Whilst functional study points
to the presence of
CFTR in the developing germ cells,
their existence awaits
confirmation by analysis of CFTR mRNA using RT-PCR and CFTR protein
by Western blot analysis.
Figure
4. CFTR chloride current in rat germ cells. Time course of cAMP-evoked
whole-cell current in germ cells (A). Membrane potential was held at -70
mV. 100 M cAMP was
applied to the pipette (internal) solution containing 120 mM CsCl and
20 mM TEA-Cl. The external solution was Krebs-Henseleit (K-H) solution.
The I-V characteristics of the cAMP-evoked current (B). The chloride
current was voltage-independent (C).
Lonidamine[59-61],
AF2785[62] and analogues have been shown to have antispermatogenic
activity. When given to rats they caused a premature shedding of spermatozoa
from the testis[63]. Although these effects have been attributed
to a disruption of germ cellSertoli cell junctional complex, their action
on germ cell CFTR cannot be excluded. While the role of the germ cell
CFTR in spermatogenesis remains unknown, it is conceivable that the antifertility
effects of the indazole compounds may in part be due to an inhibition
of germ cell CFTR.
Given the widespread role of this membrane protein in male reproduction[38],
screening of testis-specific
CFTR inhibitors can provide a new avenue of research into the development
of novel male contraceptives.
9
Acknowledgements
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Correspondence
to: Professor P.Y.D. Wong, Department of Physiology, The Chinese
University of Hong Kong, Shatin, N.T., Hong Kong.
Fax: +852-2603 5022
E-mail: patrickwong@cuhk.edu.hk
Received
2000-02-21 Accepted 2000-03-02
