Molecular and Cellular Endocrinology 250 (2006) 128–136
Odorant receptors and olfactory-like signaling mechanisms
in mammalian sperm
Marc Spehr a,∗ , Katlen Schwane b , Jeffrey A. Riffell c , Richard K. Zimmer d , Hanns Hatt b
a
Department of Anatomy and Neurobiology, Program in Neuroscience, University of Maryland
School of Medicine, Baltimore, MD 21201, USA
b Department of Cell Physiology, Ruhr-University Bochum, D-44780 Bochum, Germany
c ARL Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA
d Department of Ecology and Evolutionary Biology, Neuroscience Program, and Brain Research Institute, University of California,
Los Angeles, CA 90095-1606, USA
Abstract
Since their discovery in 1991, members of the odorant receptor (OR) family have been found in various ectopic tissues, including testis and sperm.
It took, however, more than a decade for the first mammalian testicular ORs to be functionally characterized and implicated in a reproductively
relevant scenario. Activation of hOR17-4 and mOR23 in human and mouse sperm, respectively, mediates distinct flagellar motion patterns and
chemotactic behavior in various bioassays. For hOR17-4, receptor function and downstream signal transduction events are shown to be subject to
pharmacological manipulation. Further insight into the basic principles that govern sperm OR operation as well as into the molecular logic that
underlies OR-mediated signaling could set the stage for pioneering future applications in procreation and/or contraception.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Odorant receptor; Chemotaxis; Signal transduction; Adenylate cyclase
1. Introduction
The mystery of fertilization has captivated generations of
researchers for more than a century, yet our knowledge of
many aspects of this fundamental biological process has largely
remained rudimentary (reviewed in Vacquier, 1998). As a first
step, a million-strong armada of genetically unique sperm cells,
readily equipped for the limited purpose of successfully delivering their genetic information to the egg cell, start out on
a long journey to locate their target deep inside the female
genital tract. This trek represents an enormous navigational
challenge to the smallest cells of the human body. To compensate for these difficulties, spermatozoa have developed distinct chemosensory capabilities to scan their environment and
allow spatial orientation. Candidate sperm attractants are found
widely in aquatic as well as in terrestrial organisms (Riffell
et al., 2002). In internal fertilizers, however, the nature and
source of such potential chemical guideposts as well as the
molecular mechanisms underlying chemically induced changes
∗
Corresponding author. Tel.: +1 410 706 8921; fax: +1 410 706 2512.
E-mail address: [email protected] (M. Spehr).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2005.12.035
in sperm motility are controversial questions. In this context,
an unexpected group of receptor proteins has recently taken
center stage—members of the odorant receptor (OR) family.
Conventionally found on ciliary membranes of nasal olfactory
sensory neurons (OSNs), the list of potential OR tasks is now
significantly extended as various research groups provide evidence that implicates mammalian ORs in sperm–egg chemical
communication.
2. Olfactory signaling—a blueprint for chemodetection
in ectopic systems?
The chemical senses, i.e. smell and taste, are ancient sensory
modalities that have evolved massive repertoires of receptors to
detect and discriminate molecules of immense structural variety.
Despite considerable functional differences, all chemosensory
receptor proteins currently known belong to the superfamily of
G protein-coupled receptors (GPCRs). Receptor activation triggers complex biochemical signaling cascades that transduce the
chemical energy of ligand binding into ion fluxes and resultant
changes of membrane potential. Several reports on expression
of certain chemosensory receptors in rather unorthodox tissues
have led to the assumption that these receptors, in a general
M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
sense, represent excellent candidates for high affinity chemodetectors in cells outside the nose.
2.1. Odorant receptors
1991 marked the beginning of the molecular era in chemosensory research. The landmark discovery of the rat OR gene family
by Linda Buck and Richard Axel (honored by the Nobel Committee in 2004) jump-started the field of olfaction (Buck and
Axel, 1991) and created a new framework for multi-level studies
that challenged longstanding dogmas and gradually unraveled
many mysteries of olfactory physiology. Yet, the complex functional diversity among the OR family still conceals various
surprises.
With 1.4 – 4% of all mammalian genes devoted to encoding
ORs (Mombaerts, 2004a) the physiological significance of these
receptors becomes evident. In humans, however, two-thirds of
all OR genes show sequence disruptions (an apparently random process of pseudogene formation), leaving us with about
350 functional receptors. With the exception of 20 and Y, all
human chromosomes harbor OR genes which are mostly organized in clusters (Glusman et al., 2001). These genes have served
as powerful tools in understanding the organizational logic of
the olfactory system but researchers still struggle to functionally
characterize individual ORs. In fact, just a handful of cognate
receptor–ligand pairs have been reported to date (reviewed in
Mombaerts, 2004a) and only two human ORs could be matched
to specific ligands (Wetzel et al., 1999; Spehr et al., 2003).
Numerous laboratories have been puzzled by the difficulties of
functional OR expression in heterologous cell systems. Recombinant ORs are frequently trapped in intracellular compartments
and hardly translocated to the plasma membrane (McClintock
and Sammeta, 2003). However, identification of OSN-specific
accessory proteins that appear to be involved in OR targeting (Saito et al., 2004) has again raised hopes of researchers
to accomplish a complete description of species-specific OR
repertoires in the future. Despite the few consolidated findings currently available, the prevailing model of peripheral odor
detection describes sensitivity to a variety of structurally similar ligands by individual broadly tuned ORs and, vice versa,
concentration-dependent recognition of single odorants by multiple receptors. This combinatorial code (Malnic et al., 1999)
underlies processing of odor information by spatiotemporal
activity maps in the olfactory bulb (Mombaerts et al., 1996;
Wang et al., 1998).
2.2. Olfactory signal transduction
Since the 1980s, sophisticated physiological and biochemical techniques, later combined with transgenic animal models, have shed light on the molecular mechanisms that underlie signal transduction in mammalian OSNs (for a detailed
overview see Firestein, 2001). Canonical signaling proteins are
densely packed in ciliary processes that protrude into the mucus
layer covering the olfactory epithelium. Ligand binding triggers a change in heptahelical OR conformation that activates a
membrane-bound type III adenylate cyclase (mAC III) via an
129
olfactory-specific G protein subunit (G␣olf ). cAMP-dependent
opening of cyclic nucleotide-gated (CNG) channels and successive activation of Ca2+ -gated Cl− channels results in a depolarizing receptor current that is transformed into axonal trains of
action potentials—the universal language of the central nervous
system. The outlined signaling cascade is well established for
the vast majority of OSNs. An early proposed phosphoinositide pathway (Boekhoff et al., 1990) has recently been shown to
play a rather modulatory role (Spehr et al., 2002). However, successively discovered subpopulations of less well-characterized
sensory neurons in the nose (discussed in Zufall and Munger,
2001) have added another layer of complexity to the problem of
olfactory signal transduction.
2.3. Ectopic OR expression
A growing body of evidence suggests that OR expression
is not tightly restricted to sensory neurons in the nose. Over
the years, ectopic OR transcripts have been found in a variety of tissues, including myocardial and erythroid cells (Drutel
et al., 1995; Feingold et al., 1999), ganglia of the autonomic
nervous system (Weber et al., 2002), pyramidal neurons in the
cerebral cortex (Otaki et al., 2004), as well as the spleen, brainstem, colon, and prostate (Blache et al., 1998; Raming et al.,
1998; Conzelmann et al., 2000; Yuan et al., 2001). As each of
these receptors has been identified using conventional molecular amplification techniques and none has yet been attributed
a physiological function, considerable caution has to be used
when assigning a functional meaning to these findings.
Only 1 year after the breakthrough discovery of the rodent
OR gene family, a Belgium group led by Marc Parmentier
(Parmentier et al., 1992) demonstrated transcription of about
20 mammalian OR genes in cells of the male germ line,
most notably during late stages of spermatogenesis. Followup studies employed polyclonal antibodies to localize specific ORs to the midpiece and base of the flagellum of
mature sperm (Vanderhaeghen et al., 1993; Walensky et al.,
1995). Using RNase protection assays, Parmentier’s group later
reported predominant but not exclusive testicular expression
of some of these ORs (Vanderhaeghen et al., 1997a) and
estimated a total of up to 50 ectopically expressed receptors
(Vanderhaeghen et al., 1997b). Similar numbers were recently
confirmed in a high-throughput oligonucleotide microarray
approach that detected 66 OR genes enriched in mouse testis
(Zhang et al., 2004). Strikingly, testicular ORs cannot be classified as members of a specific OR subfamily based on common sequence characteristics (Vanderhaeghen et al., 1997b).
However, the average level of amino acid conservation as
well as the percentage of intact sequences among testicular
ORs is reported to be higher than in ordinary nasal receptors
(Branscomb et al., 2000), strongly indicative of a physiological function. Deciphering the biological role of OR expression in developing and mature sperm cells has thus become
a major interdisciplinary challenge that brings together neuroscientists, reproduction and developmental biologists, basic
as well as clinical researchers (discussed in Spehr and Hatt,
2004).
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M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
3. Sperm and internal fertilization
From marine broadcast spawners to higher terrestrial vertebrates (including humans), the basic principles of fertilization
have been largely conserved. In a remarkably profligate process,
several hundred millions of sperm cells are ejaculated to locate
the egg either in the turbulent ocean or the relatively benign
mammalian reproductive tract. In humans, however, only a small
fraction of spermatozoa eventually make their way to the fertilization site, the oviductal ampulla (reviewed in Eisenbach,
1999).
3.1. Tiny size, complex physiology
Sperm size and morphology considerably varies across
species. In mammals, spermatozoa are singularly small, highly
polarized, hydrodynamically shaped motile cells that are composed of a head (2–5 ␮m in diameter) containing the condensed
nucleus and acrosome, a mitochondria-rich midpiece, and an
adjacent tail that mainly consists of one central axoneme. While
immotile during testicular maturation and epididymal transit,
ejaculated spermatozoa exhibit distinct tail beating patterns and
swimming behaviors that are based on pulsative axoneme filament contractions governed by intracellular Ca2+ waves (Luconi
and Baldi, 2003). In a loosely characterized maturation process collectively termed capacitation, sperm undergo a predetermined series of biochemical and functional changes inside
the female genital tract. As only capacitated sperm are able to
exocytotically release a set of proteolytic enzymes from their
acrosome in order to penetrate the egg’s glycoprotein coat, the
zona pellucida, this post-ejaculatory maturation process is a crucial prerequisite for successful fertilization (Yanagimachi, 1994;
Jaiswal et al., 1999a).
Surprisingly, a number of receptors, enzymes, and ion channel proteins originally regarded as neuron-specific have recently
been described in sperm (discussed in Darszon et al., 1999;
Meizel, 2004). Some of those are well-described members of
both visual and olfactory GPCR-mediated signal transduction
pathways [G proteins (Baxendale and Fraser, 2003a), G protein receptor kinase 3 and ␤-arrestin 2 (Walensky and Snyder,
1995), cone photoreceptor CNG channels (Weyand et al., 1994;
Wiesner et al., 1998), particulate adenylate cyclases (Defer et
al., 1998; Gautier-Courteille et al., 1998; Baxendale and Fraser,
2003b)]. Given the concomitant expression of a subset of ORs in
mammalian sperm, it is tempting to speculate that some of these
sensory cascade proteins could link OR activation to intracellular Ca2+ waves and, thus, significant changes in flagellar beating
and motility. However, mainly due to a puzzling immanent difficulty to directly record ionic currents from sperm cells using
the patch-clamp technique (Darszon et al., 2004), investigation
of “neuron-like” signaling events in sperm is still in its infancy.
3.2. Two opposing concepts—random gamete collision
versus chemical guidance
Chemotaxis is defined as directed movement with respect to
a chemical concentration gradient. In sharp contrast to marine
invertebrates, the biological relevance of mammalian sperm
chemotaxis has long been a matter of debate. In recent years,
however, accumulating evidence has established the occurrence
of mammalian chemotaxis in vitro beyond doubt (VillanuevaDiaz et al., 1992; Ralt et al., 1994; Cohen-Dayag et al., 1995;
Eisenbach, 1999; Jeon et al., 2001; Fabro et al., 2002; Sun
et al., 2005). Given the tortuous path sperm has to travel to
reach the oviductal ampulla as well as the disproportionate
dimensions of the female genital tract, successful fertilization
based on a concept of random gamete collision appears largely
unlikely. On its way to the ampulla, sperm passes the cervix and
uterus (70–80 mm), locates and enters the opening of the oviduct
(20–50 ␮m in diameter), travels along the tube (50–80 mm), and
finally passes the oviductal isthmus, a mucus-filled constriction
of the tube (Eisenbach and Tur-Kaspa, 1999). Prior to ovulation, sperm are frequently decelerated and bound to the tubal
epithelium (Barratt and Cooke, 1991; Suarez, 1998). Both the
timing of sperm capacitation and their release from oviductal
storage sites appear to be programmed according to egg availability (Giojalas et al., 2004). Moreover, the ovulatory ampulla
contains a significantly larger proportion of spermatozoa than
found in the contralateral tube (Williams et al., 1993). These
findings strongly implicate sperm in a dynamic dialogue with its
environment and/or the egg cell itself. The concept of selective
recruitment and sperm navigation aided by chemical guidance
cues has recently been supplemented by the idea of long-range
pathfinding due to a tubal temperature gradient (Bahat et al.,
2003). However, both the chemical identity of potential attractive
components in female body fluids and the exact timing and location of their release remain mysterious. A number of candidate
attractants (heparin, progesterone, acetylcholine) have failed
careful examination (Eisenbach, 1999; Jaiswal et al., 1999b).
Interestingly, recent data suggest that both the oocyte and cells of
the surrounding cumulus oophorus independently secrete sperm
chemoattractants (Sun et al., 2005) and the principal function of
these soluble factors might rather be in increasing egg target
size than in acting as a gate to minimize/eliminate hybridization
(Riffell et al., 2004). ORs as mediators of sperm–egg communication therefore represent an attractive model that has only
recently been experimentally addressed. Data currently available fuels the debate about a significant in vivo function of these
unconventional sperm receptors in male fertility.
4. Odorant receptors and sperm motility
Identification and functional characterization of both a human
(Spehr et al., 2003) and a mouse (Fukuda et al., 2004) testicular
OR represent major steps toward a deeper understanding of the
role ORs play in mammalian sperm. Both receptors, hOR174 (synonymous OR1D2) in humans and mOR23 (synonymous
mOR267-13) in mice, are activated by small aldehyde molecules
and mediate robust Ca2+ signals in mature sperm. Employing
multi-level investigational approaches, the physiological functions of these receptors are now being uncovered.
hOR17-4 transcripts have initially been amplified by RT-PCR
from biopsates of human testicular tissue (Spehr et al., 2003),
and surface expression of the receptor on mature sperm has
M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
subsequently been confirmed by proteomic analysis using Multidimensional Protein Identification Technology (MudPIT; Spehr
et al., 2004b). As previously shown for a number of conventional ORs (Wellerdieck et al., 1997; Wetzel et al., 1999, 2001),
recombinant expression of chimeric and full-length hOR17-4
clones in a heterologous HEK293 cell line allowed for identification and structure–function analysis of cognate receptor–ligand
pairs using ratiofluorometric single cell calcium recordings.
Since this venture is best described by the image of the needle
in the haystack, receptor activation by complex odorant mixtures was initially tested, then active odor fractions were further
subdivided. This strategy ultimately identified cyclamal and its
structural analog bourgeonal as potent activators of recombinant
hOR17-4. Based on cyclamal as a template, selective feature
modifications outlined the receptor’s molecular receptive range.
hOR17-4 accommodates small aromatic aldehydes of defined
length with bourgeonal acting as the most potent ligand tested
so far. This compound is a common synthetic perfume ingredient that mimics the scent of lilies of the valley. Surprisingly, the
relatively simple aliphatic aldehyde undecanal competes for the
receptor’s ligand binding pocket without activating hOR17-4.
This inhibitory effect appears receptor specific as another aldehyde sensitive OR of the same gene cluster (hOR17-40; Wetzel
et al., 1999) proved unaffected by undecanal. We are just beginning to understand the principles of antagonistic OR regulation
(Spehr et al., 2003, 2004a; Oka et al., 2004), adding this largest
subpopulation of GPCRs to the growing group of potential pharmacological targets.
Any conclusions drawn from recombinant expression analysis require functional validation in the natural system—in this
case human sperm. In both single cell Ca2+ imaging recordings and ratiofluorometric population screenings, bourgeonal
induced robust and dose-dependent Ca2+ fluxes over the plasma
membrane in a considerable proportion of sperm (Spehr et al.,
2003). The odor sensitivity spectrum of sperm largely mirrors
the receptive field of recombinant hOR17-4. In sperm, however, the response thresholds recorded are remarkably lower.
Moreover, the inhibitory effect of undecanal is not an artefact of recombinant receptor expression. Dependent on relative
agonist/antagonist concentrations, undecanal potently blocks
sperm activation by bourgeonal, confirming a functional role
of hOR17-4 in at least a fraction of mature sperm.
Does hOR17-4 govern any significant sperm behaviors?
Knowing the receptor’s activation signature now provides a tool
to investigate its role in sperm motility and chemical sperm–egg
communication. A combination of microcapillary accumulation assays and video motion analysis of swimming sperm
revealed strong and dose-dependent chemotaxis and chemokinesis (enhanced swimming speed; Spehr et al., 2003) as well as
hyperactive flagellar beating (Spehr et al., 2004b) in presence of
an ascending bourgeonal gradient (Fig. 1). These motility patterns, however, were completely abolished by coapplication of
undecanal. Strikingly, the proportion of chemotactically guided
sperm in presence of bourgeonal is similar to the fraction of cells
that is activated by oocyte-cumulus conditioned media (Sun et
al., 2005). It, thus, appears likely that navigating sperm cells
utilize hOR17-4 and/or further ORs to detect chemical guid-
131
ance cues and translate this information into stereotyped motion
patterns.
In parallel with work on humans, Fukuda et al. (2004) recently
introduced the power of mouse transgenic approaches to the
problem of OR expression in sperm. Transcripts of a distinct
mouse receptor, mOR23, have been found in the olfactory
epithelium as well as testicular tissue (Asai et al., 1996). Lyral,
another floral aldehyde odorant, specifically activated mOR23 in
both homologues and heterologous expression systems (Touhara
et al., 1999). The compound also induced Ca2+ transients in
a fraction of mouse spermatogenic cells and mature spermatozoa (Fukuda et al., 2004). Sperm derived from transgenic
animals that overexpressed mOR23 under the control of a testisspecific promoter showed increased Ca2+ signals and navigated
along ascending lyral gradients. Thus, OR-controlled swimming
behavior appears to be a general phenomenon in mammals.
Interestingly, the mouse hOR17-4 ortholog is a pseudogene,
explaining the lack of bourgeonal-induced Ca2+ responses in
mouse sperm (Fukuda et al., 2004) and suggesting a speciesspecific repertoire of reproductively relevant ORs.
5. OR-induced signaling pathway(s) in sperm
Uncovering the mechanistic link between OR activation and
characteristic changes in flagellar beating poses a profound scientific challenge. Expression analysis of candidate signal transduction proteins by conventional mRNA-based approaches (i.e.
in situ hybridization or RT-PCR) is inapplicable to mature sperm
as these cells have shut down their protein synthesis machinery.
MudPIT offers a solution to this problem. Combining twodimensional liquid chromatography and tandem mass spectrometry, this “shotgun” proteomics approach identified expression
of various stimulatory G␣ -proteins, including Golf , as well as
all nine mAC isoforms in human sperm (Spehr et al., 2004b).
Immunocytochemistry located Golf and mAC isoforms III and
VIII to distinct regions on the tail and/or the midpiece, exactly
where flagellar beating is initiated. Careful spatiotemporal analysis of bourgeonal-induced responses in immobilized sperm
further demonstrates that Ca2+ signals originate in the midpiece.
In pharmacological tests, these responses as well as bourgeonalmediated swimming behaviors were abolished by specific P-site
inhibitors of mACs (Fig. 1), proposing a mechanistic model for
hOR17-4 signaling in human sperm that exhibits striking similarities to the olfactory transduction pathway. This notion is
further supported by a recent report that implicates mAC III in
normal sperm function and male fertility in mice (Livera et al.,
2005). However, the identity of downstream effectors of cAMP,
the product of mAC activity, has yet to be elucidated. Currently,
both direct gating of Ca2+ -permeable ion channels and activation
of intermediary cAMP-dependent enzymes represent conceivable options.
6. Bifunctionality of sperm ORs
Whether sperm ORs are restricted to reproductive functions
or additionally perform their “conventional” task in olfaction
is a longstanding question. In favor of a dual function model,
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M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
Fig. 1. Swimming behavior of human sperm is dramatically changed in response to bourgeonal, a cognate ligand of the testicular olfactory receptor hOR17-4 (from
Spehr et al., 2003, 2004b). (A) Placed in an ascending gradient of (A1 ) 10−9 to 10−5 M bourgeonal [human tubal fluid (HTF) as control], or (A2 ) mixtures that
combined 10−7 M bourgeonal with 10−9 , 10−8 , or 10−7 M undecanal, sperm exhibit strong and dose-dependent chemotactic as well as chemokinetic effects which
are inhibited by undecanal at relative concentrations ≥0.1:1.0. Open circles correspond to video images captured at intervals of 0.033 s with arrowheads indicating the
direction of travel of randomly selected sperm. Complete data sets were used to calculate the mean (±S.E.M.) swimming speeds, angles (θ), and vector lengths. The
angle of sperm orientation is defined as the shortest tangent between each cell and the capillary tip. A vector length of 1 indicates all cells swim in a common direction
while a vector length of 0 indicates random motion. (B) Accumulation of human sperm in microcapillary tubes presenting either an ascending, uniform, or descending
bourgeonal concentration gradient. Cell densities significantly increase in capillaries presenting an ascending gradient, a result confirming a chemotactic mechanism.
(C and D) A specific P-site inhibitor of mAC activity (SQ22536) affects sperm behavioral response to bourgeonal. (C) Accumulation of sperm in microcapillary
tubes. (D) Flagellar beat frequency of cells swimming within 100 ␮m of the capillary tip. (E) Sperm swimming behavior imaged near a capillary tip. Top panel, left:
representative sperm path demonstrating a chemotactic turn having detected a decrease in bourgeonal concentration. Top panel, right: seven sequential frames of
digital video images corresponding to the closed circles in the path. Highlighted are video frames that show the asymmetrical flagellar bending during a chemotactic
turn. Middle panel: sperm exhibit no chemotactic turning in presence of 10 mM SQ22536. Bottom panel: sperm swim without rapid turns when challenged with HTF.
(F and G) Average percentage of total sperm entering or leaving microcapillary tubes over 30 s recording intervals. (H) Of those sperm leaving the capillary tube,
the percentage of which turn around and re-enter the termini. Each plotted value is a mean (±S.E.M.). For (C), (D), (F), and (G), an asterisk indicates a significant
difference between test and control stimuli for each incubation treatment (Scheffé test: * p < 0.05 and *** p < 0.001).
mOR23 had originally been identified from lyral-sensitive OSNs
(Touhara et al., 1999). Likewise, RT-PCR using intron-spanning
primers and subsequent Southern blot analysis revealed expression of hOR17-4 in biopsates of human olfactory epithelium
(Spehr et al., 2004a). Although definite proof is lacking, psychophysical studies and electro-olfactogram recordings on a
large number of human subjects suggest a basically identical
receptor operation in both olfactory tissue and spermatozoa. This
idea of bifunctional ORs (Fig. 2) could provide a framework for
comparative future analysis of receptor expression, function and
regulation in both systems. It remains to be seen if any single
nucleotide polymorphisms (Olender et al., 2004) in the hOR174-encoding gene are reflected in modified olfactory perception
of bourgeonal-like odorants and/or potential defects in sperm
behavior. Although purely speculative, future diagnosis of sperm
receptor malfunction may be feasible via simple sniffing tests.
M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
133
Fig. 2. Proposed model of a bifunctional human odorant receptor. Either expressed in ciliary membranes of nasal OSNs or on the midpiece of mature sperm,
hOR17-4 (OR1D2) is activated by the synthetic floral odorant bourgeonal and competitively inhibited by undecanal. In both systems, receptor activation triggers
a cAMP-dependent signaling cascade. In sperm, however, the identity of the G protein and mAC isoform involved as well as the nature of downstream signaling
components remains elusive. (A–C) Bourgeonal-induced signals in human olfactory epithelium and sperm. (A) Electro-olfactogram recordings from the olfactory
mucosa show considerable inhibition of bourgeonal-induced field potentials after brief undecanal exposure. (B and C) Ca2+ signals in individual sperm are blocked
by coapplication of both undecanal (B) and SQ22536 (C).
7. Perspectives
After years of controversial debate, functional characterization of the first testicular ORs in humans and mice, respectively,
should provide long-awaited tools to unravel the mysteries of
OR expression in mammalian sperm. In a collaborative effort,
various groups now extend their research to this field, legitimating speculation about potential future applications in reproductive medicine. Transferability of current in vitro findings to
the in vivo situation, however, awaits conclusive proof. Thus,
the recent discoveries can only be regarded as a first building
block toward a general understanding of OR-mediated sperm
physiology.
Human as well as mouse sperm respond to OR activation with
a number of distinct motility changes. Directed movement along
ascending stimulus concentrations, dramatically elevated swimming speeds, hyperactive flagellar beating, and characteristic
“flip-turns” upon detection of a decrease in stimulus concentration (Spehr et al., 2004b) demonstrate the versatility of behavioral responses (Fig. 1). Whether the functional significance of
sperm ORs becomes manifest in direct sperm–egg communication or if ORs participate in other pre-fusion processes that
prime sperm for fertilization (e.g. induction of capacitation or
the acrosome reaction, release from oviductal storage, etc.) will
be addressed in future research. Narrowing down the secretion
site(s) of potential chemical guidance cues to either the egg
itself, the surrounding cumulus cells, or even endothelial cells
of the tube, uterus or cervix will be a major step toward that
goal. Clearly, bourgeonal, cyclamal, or lyral do not represent
actual endogenous OR ligands emitted inside the female body.
These synthetic compounds can only be regarded as molecular templates that reflect the apparent structural requirements
for an endogenous signaling molecule. On-going studies focus
on content analysis of different bioactive female body fluids
in search for any constituents that meet such specifications.
Once identified, this knowledge will provide a basis for the synthetic design of specific and even more potent pharmacological
analogs.
Estimated numbers of testicular ORs in mammals range
between 20 and 66 (Vanderhaeghen et al., 1997b; Zhang et al.,
2004). However, we still lack a clear picture of their cellular
distribution or their expression profile during different developmental stages. Aside from hOR17-4 and mOR23 which function
in mature sperm, other testicular ORs could additionally play
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M. Spehr et al. / Molecular and Cellular Endocrinology 250 (2006) 128–136
pivotal roles in spermatogenesis or epididymal maturation. In
this context, one line of future research will examine whether
the complete repertoire of testicular ORs is expressed by each
sperm cell or if individual expression patterns designate distinct sperm subpopulations. On the other extreme, each mature
sperm cell may only express one particular OR, a situation
reminiscent of tightly controlled receptor expression in OSNs.
Numerous studies on this physiological rarity have given rise to
the widely popular ‘one neuron–one receptor’ model (Lewcock
and Reed, 2004; Serizawa et al., 2003), although it has been
convincingly argued that this hypothesis is still far from being
proven (Mombaerts, 2004b; Spehr and Leinders-Zufall, 2005).
If, however, individual spermatozoa express different ORs, this
could have profound functional implications. Particular receptors might be used in different behavioral contexts, enabling
sperm to adequately respond to a variety of cues on their journey to the egg.
The key role of particulate adenylate cyclase(s) in sperm ORmediated signaling contrasts the widespread view of a soluble
bicarbonate-activated adenylate cyclase as the predominant or
even exclusive cAMP-synthesizing enzyme in sperm (Chen et
al., 2000). However, Ca2+ -dependent crosstalks between both
types of cyclases cannot be excluded. The effective target
of local cAMP increases within the midpiece and flagellum
remains elusive (Fig. 2). The cone-type CNGA3 channel subunit found in sperm (Weyand et al., 1994; Wiesner et al., 1998)
apparently prefers cGMP to cAMP and the potentially cAMPactivated CatSper1 and CatSper2 channels (Ren et al., 2001;
Quill et al., 2001; Carlson et al., 2003) still lack detailed functional description. Moreover, activation of secondary cAMPdependent enzymes (e.g. PKA, etc.) could additionally be
involved.
Pharmacological modulators of sperm ORs or specific mAC
isoforms could have considerable therapeutic benefit, e.g. by
improving the success rate of in vitro fertilization (IVF) treatment, a process still far from being considered satisfactory
(Spehr and Hatt, 2005). Sperm ORs could additionally provide
promising pre-fusion targets in new approaches to hormone-free
contraception. However, such speculation may still be a long way
from future drug development and subsequent clinical trials. It
will be challenging to demonstrate efficiency and inoffensiveness of potential pharmaca as well as to discover suitable ways
of drug application.
Acknowledgements
Our work on OR-mediated signaling in sperm is supported by
the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (M.S.), by the Heinrich and Alma Vogelsang Foundation
(K.S.), by NSF awards IBN 01-32635 and IBN 02-06775, as well
as grants from the UCLA Council on Research, and NOAA California Sea Grant College Program R/F-197 (R.K.Z.), and by the
University of Arizona Center for Insect Science NIH training
grant No. 1K126M00708 (J.A.R.).
We thank Thomas Lichtleitner for preparing figures and illustrations, Harald Bartel for excellent technical support, and Jasmin Gerkrath for technical assistance.
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