Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 761-767
doi:10.1111/j.1745-7270.2008.00453.x
Identification and expression of GABAC receptor in rat testis and spermatozoa
Shifeng Li#, Yunbin Zhang#, Haixiong Liu, Yuanchang Yan, and Yiping Li*
Laboratory of Molecular Cell Biology, Shanghai
Key Laboratory for Molecular Andrology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031,
China
Received: April 3,
2008 Accepted: May 4,
2008
This work was
supported by grants from the Major State Basic Research Development Program of
China (2007CB947100) and the Shanghai Municipal Commission
for Science and Technology (074319111 and 07DZ22919)
#These authors
contributed equally to this work
*Corresponding
author: Tel, 86-21-54921395; Fax, 86-21-54921415; E-mail, [email protected]
Our previous studies showed that g-aminobutyric acid (GABA)A and GABAB receptors are involved in rat sperm acrosome reaction
induced by progesterone or GABA. Here, we report the presence of GABAC receptor in rat testis and spermatozoa.
Full-length complementary DNA encoding the r1, r2 and r3
subunits of GABAC receptor
were cloned from rat testis; their sequences are identical to those of rat GABAC receptor in retina. Reverse
transcription-polymerase chain reaction analysis showed that during the
development of rat testis, the transcript levels of the r1 and r2 subunits showed little change, while the expression
of r3 was gradually up-regulated.
Immunofluorescence analysis using an anti-r1 antibody revealed that GABAC receptor exists on the elongated spermatid
and sperm. Using a chlortetracycline assay, we found that
N(4)-chloroacetylcytosine arabinoside, a GABAC receptor agonist, triggered rat sperm acrosome
reaction; whereas (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid, a GABAC receptor antagonist, inhibited the ability
of N(4)-chloroacetylcytosine arabinoside to induce acrosome reaction. These
results suggested that GABAC receptors are also involved in rat sperm acrosome reaction.
Keywords GABAC receptor; g-aminobutyric
acid; N(4)-chloroacetylcytosine arabinoside; spermatozoa; acrosome reaction
The g-aminobutyric acid (GABA) is the main inhibitory transmitter in the
mammalian central nervous system. It exerts its effects through three distinct
classes of membrane receptors: GABAA, GABAB and GABAC. GABAA and
GABAC receptors are ligand-gated ion channels, while GABAB receptors are G-protein-coupled receptors. GABAC receptors appear to be much simpler than GABAA receptors, but they have not been studied as extensively as GABAA receptors [1]. GABAC receptors are more sensitive
to GABA than GABAA receptors [1]. GABAC
receptors are not blocked by bicuculline, a selective antagonist of GABAA receptors, and are not activated by baclofen, an agonist of GABAB receptors [2]. The r subunits of GABAC receptors are capable of
forming functional homo-oligomeric or hetero-oligomeric receptors, whereas GABAA receptors are efficiently expressed only as hetero-oligomers [3].
Two subunits (r1 and r2) have been cloned from humans, whereas three subunits (r1–3) have been
cloned from rats. The ? subunits have been detected in the retina,
thalamus, hippocampus, pituitary and gut [4–9]. They may play roles in
visual processing, regulation of sleep-waking rhythms, pain perception, memory,
learning, regulation of hormones and neuroendocrine gastrointestinal secretion.The mammalian sperm is referred to as a neuron with a tail, due to
the presence of “neuronal” receptors in the sperm plasma membrane and
because some of the reported receptors are related to physiological activities
of mammalian spermatozoa, including sperm motility, capacitation and acrosome
reaction [10]. The GABAergic system exists in various somatic tissues [11,12].
At relatively low concentration, GABA can mimic the effects of progesterone by
promoting acrosome reaction of capacitated human and mouse spermatozoa [13,14].
The rat oviduct contains twice the amount of GABA as found in rat brain [15].
We previously showed that GABAA receptors were detected in rat
spermatozoa and were involved in acrosome reaction triggered by GABA and
progesterone [16]. GABAB receptor transcripts were
identified in rat testis, and immunofluorescence experiments with an anti-GABABR1 antibody detected immunoreactivity in the head of rat spermatozoa
[17]. GABA-initiated acrosome reaction of the rat spermatozoa can be inhibited
by baclofen. Thus, the induction of acrosome reaction in rat sperm by GABA is
regulated by the proportionality of activated GABAA and
GABAB receptors acting as a yin-yang control [18]. The existence of
GABAC receptors in rat spermatozoa, the potential function of GABAC receptors in sperm, and their interaction with GABAA or GABAB receptors during acrosome reaction, however,
have not been examined yet.Here, we report for the first time the presence of GABAC receptors in rat spermatozoa. We also present evidence that GABAC receptors facilitate rat sperm acrosome reaction.
Materials and Methods
Materials
The following chemicals and reagents were purchased from Sigma (St.
Louis, USA): bovine serum albumin (Fraction V), b-mercaptoethanol, Triton
X-100, Tween-20, GABA, N(4)-chloroacetylcytosine arabinoside (CACA),
(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), 4‘,6-diamidino-2-phenylindole
(DAPI), and goat-anti-rabbit immunoglobulin G (IgG) conjugated to
fluorescein-isothiocynate (FITC). Three-month-old Sprague Dawley rats were
obtained from Shanghai Laboratory Animal Company (Shanghai, China). Avian
myeloblastosis virus (AMV) reverse transcriptase was purchased from Promega
(Madison, USA). Trizol was purchased from Invitrogen (Carlsbad, USA). Anti-GABAC receptor r1 subunit antibody was purchased from Santa Cruz Biotechnology
(Santa Cruz, USA). Goat-anti-rabbit IgG conjugated to horseradish peroxidase
were from Kangchen Bio-Tech (Shanghai, China). Enhanced chemiluminescence
reagents were purchased from Pierce Biotechnology (Rockford, USA).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
To examine the expression pattern of GABAC receptors
at different stages of spermatogenesis, total RNA obtained from rat testis (at
the age of 1, 5, 10, 20, 30, 40, 60 and 90 d) were used for RT. Total RNA
extraction was carried out using Trizol reagent according to the manufacturers
protocol, and complementary DNA (cDNA) was synthesized by oligo(dT) primers
with AMV reverse transcriptase. Gene-specific primers designed to amplify each
cognate region are listed in Table 1. Amplification was carried out for
30 cycles using the following conditions: 94 ?C for 30 s; 55 ?C, 58 ?C or 60 ?C
(annealing temperature for the r1, r2 and r3 subunits, respectively) for 30 s; and 72 ?C for 1 min. Primers for
b-actin as a control were employed as follows: forward primer 5‘-AACGAGCGGTTCCGATGCCCTGAG-3‘
and reverse primer 5‘-TGTCGCCTTCACCGTTCCAGTT-3‘.
Preparation of protein samples
Spermatozoa were collected from caudal epididymis and washed twice
in phosphate-buffered saline (PBS). Testis, retina and spermatozoa were
homogenized in Preparation of modifed radioimmunoprecipitation lysis buffer [1?PBS, 1% Nonidet P-40, 0.1% sodium dodecylsulfate (SDS), 5 mM EDTA,
0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl
fluoride], vortexed for 1 h and centrifuged at 12,000 g for 10 min at 4
?C. The supernatant was collected and considered as protein extract of the
respective tissues. Samples were directly suspended in 2?SDS sample buffer and boiled for 5 min. Lysates were centrifuged at
12,000 g for 10 min. The supernatant fractions were used for Western
blot analysis.
Western blot analysis
Protein samples were separated by electrophoresis on a 10% SDS
polyacrylamide gel. After electrophoresis, the separated proteins were transferred
onto a nitrocellulose membrane and blocked with 5% dry milk in Tris-buffered
saline with 0.1% Tween-20. The blotted membrane was incubated with anti-r1 antibody at 4
?C overnight. The membrane was washed three times with Tris-buffered saline with
0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated
goat-anti-rabbit IgG secondary antibody at room temperature for 2 h. The
membrane was washed and visualized using enhanced chemiluminescence agents.
Immunofluorescence analysis
Immunofluorescence staining was carried out using the indirect
FITC-conjugated method. Rat testis cryosections (10 mm thick) were postfixed in
4% paraformaldehyde (W/V) and air-dried. Spermatozoa were fixed
on slide glass in 4% paraformaldehyde. Slides were permeabilized with 0.1%
Triton X-100 in PBS at room temperature for 15 min followed by incubation with
3% bovine serum albumin for 30 min to block non-specific reaction.
Subsequently, slides were incubated with anti-r1 antibody (1:100 dilution)
overnight at 4 ?C. After three washes, they were incubated with FITC-labeled
goat-anti-rabbit IgG (1:400 dilution) for 1 h and washed three times to remove
any unbound antibody. The stained samples were observed under an Olympus BX51
fluorescence microscope (Tokyo, Japan).
Collection, incubation, and treatment of spermatozoa
Rats were killed by cervical dislocation. The caudal region of
epididymidis was prepared free of fat and capillaries. Spermatozoa were
released from the caudal epididymidis and suspended in a modified medium [16].
The sperm concentration was estimated by using a hemocytometer and adjusted to
a final density of 1.0?107 cells/ml. After incubation at 37 ?C under 5% CO2 for 3 h, spermatozoa were then exposed to different stimuli for 20
min. Antagonists were added 5–10 min prior to the treatment with agonists. Aliquots of sperm
suspension were processed for chlortetracycline staining.
Chlortetracycline (CTC) assay for acrosome reaction
The staining procedure was modified based on the previously reported
CTC staining method [16]. CTC staining solution (1.5 mM) was freshly prepared
using a buffer (pH 7.8) containing 20 mM Tris, 130 mM NaCl and 5 mM L-cysteine.
The solution was shielded from light at room temperature. After the treatment
with agonist and/or antagonist, 200 ml aliquot of sperm suspension was mixed with
an equal volume of CTC solution in an Eppendorf tube. After 30 s, 32 ml 12.5%
paraformaldehyde in PBS (pH 7.4) was added. After gentle vortexing, 200 ml glycerol was
mixed with the sperm suspension to prevent the fluorescence from fading. More
than 200 cells per sample were examined.
Statistical analysis
Results are presented as mean±SEM throughout the study. One-way
analysis of variance of CTC staining data was performed using Microsoft (United
States) Office Excel followed by Students t-test. P<0.05 was considered to be significant.
Results
Expression of GABAC receptor in rat testis
and spermatozoa
To examine whether GABAC receptors are expressed
in rat testis, total RNA were prepared from rat testis and subjected to RT-PCR.
The PCR products were analyzed by electrophoresis on agarose gel and then
sequenced [Fig. 1(A)]. cDNA corresponding to the full-length coding
sequences of GABAC receptor subunits r1, r2 and r3 were identified,
and they were found to be identical to the published rat GABAC receptor sequences. To examine the developmental expression pattern
of GABAC receptors in rat testis, testis cDNA from 1 d postnatal to 90 d
postnatal were prepared, and expression levels of the three subunits of GABAC receptors were analyzed. The expression levels of r1 and r2 subunits
exhibited little change, but the expression level of r3 was up-regulated
gradually with the development of rat testis [Fig. 1(B)]. Western blot
analysis was used to examine the expression of GABAC
receptors in rat testis and spermatozoa. One intense band (approximately 45
kDa) was detected in rat spermatozoa sample using the anti-r1 antibody [Fig.
1(C)]. A similar band was observed in the rat retina sample which was used
as a positive control. The size of the detected r1 band is smaller than the
predicted molecular weight (approximately 55 kDa). A similar band was also
detected in the testis sample.
Localization of GABAC receptors in rat testis
and spermatozoa
The cellular localization of GABAC
receptors in rat testis and spermatozoa was visualized using immunofluorescence
microscopy. Rat testis cryosections were assayed using an antibody specific to
the C-terminus of the r1 subunit. Positive immunoreactivity was detected principally in the
region of the seminiferous tubules of the rat testis sections containing
elongated spermatozoa (Fig. 2). Immunocytofluorescence study with the
same antibody showed that the non-capacitated rat spermatozoa exhibited intense
fluorescence mainly at the anterior portion of the sperm tail, with slight
staining at the sperm head (Fig. 3). The region of the r1 signal at the
rat sperm tail appears to be mid-piece, which is reminiscent of the
localization of sperm mitochondria-associated cysteine-rich protein [19]. As a
control, no staining occurred when the primary antibody was omitted (data not
shown).
Function of GABAC receptors in rat
spermatozoa
To examine the role of GABAC receptors in rat sperm acrosome
reaction, both agonist and antagonist of GABAC
receptors were assessed for their effect using the CTC staining assay.
GABA-induced acrosome reaction in capacitated rat spermatozoa can be mimicked
by the action of CACA, a selective agonist for GABAC
receptors. As shown in Fig. 4(A), the effect of CACA was biphasic, with
a dose-dependent increase in the proportion of spermatozoa undergoing acrosome
reaction at low concentrations of CACA. The concentration of CACA producing the
highest effect was 50 mM. Further increase in CACA concentration resulted in a reduction in
stimulation. Furthermore, TPMPA [20], a GABAC
receptor selective antagonist, inhibited the induction of rat sperm acrosome
reaction by CACA [Fig. 4(B)]. TPMPA did not completely inhibit rat sperm
acrosome reaction induced by GABA.
Discussion
We have previously shown that GABAA
receptors, GABAB receptors and GABA transporter 1 (GAT1) are
expressed in rat testis and spermatozoa [16,17,2123]. It is possible that GABAC receptors also exist in male reproductive tissues. In this study,
GABAC receptors, as the component part of GABAergic system, were detected
in rat testis. The expression level of GABAC
receptors in rat testis is higher than other tissues except retina (data not
shown). The three subunits of GABAC receptors showed
different expression levels as rat testis developed. The role of GABAC receptors in differentiation and development of germ cells is
currently not understood. Geigerseder et al reported that GABA can
stimulate the proliferation of Leydig cells through GABAA receptors in vitro [24]. Because both GABAA and GABAC receptors are chloride channels, it will be
interesting to discover whether GABAC receptors are involved
in rat spermatogenesis.The distribution of GABAC receptors in rat testis
is similar to that of GABAA receptors and GAT1 [16,21].
These results suggest that GABAA and GABAC receptors are co-expressed in rat germ cells during
spermatogenesis. The location of GABAC receptors on rat
spermatozoa was different from that of GABAA
receptors, which were mainly localized on the sperm head; the location was
similar to that of GAT1, which was localized on the tail and the entire head
except for the equatorial sector [16,21]. Sperm mitochondria are limited to the
mid-piece. Mitochondrial ATP is an important source of energy for the tail
[25]. The localization of GABAC receptors at the mid-piece of
the sperm tail raises the possibility that GABAC
receptors might be involved in the regulation of sperm motility. Indeed, GABA
has been shown to affect increases in the percentage of human spermatozoa
hyperactivation [26,27]. However, these studies only assayed the involvement of
GABAA receptors. Our results provide the impetus for further research to
elucidate the role of GABAC receptors in the regulation of
sperm motility.It has been shown that GABA induces acrosome reaction in human,
mouse, and rat spermatozoa mainly through GABAA
receptors [13,16,28]. However, the role of GABAC
receptors in inducing acrosome reaction has not been examined. In rat
spermatozoa, CACA can trigger acrosome reaction that can be blocked by TPMPA.
However, TPMPA does not completely inhibit rat sperm acrosome reaction induced
by GABA. This is likely due to GABA-induced acrosome reaction being mediated by
both GABAA and GABAC receptors, and thus, selective antagonism of
GABAC receptors by TPMPA would not affect GABA signaling through GABAA receptors. This is the first study to demonstrate the involvement
of GABAC receptors in inducing acrosome reaction. The finding that both GABAA and GABAC receptors are localized on rat sperm and
involved in regulating the same physiological activity is reminiscent of the
actions of these two types of receptors in neuronal systems. In mammalian
retina, both GABAA and GABAC
receptors are localized to bipolar cell terminals [29]. Application of GABA to
neurons of the rat major pelvic ganglia produced a biphasic response, an
initial depolarization (GABAd) followed by a
hyperpolarization (GABAh). GABAA
receptors mediate the early GABA-induced GABAd,
whereas GABAC (or GABAAor) receptors mediate the late GABA-induced
late GABAd and the GABAh [30]. There is evidence
suggesting that GABAA and GABAC
subunits can form a heteromeric receptor in central neurons and that
heteromeric complexes of GABAA and GABAC receptor subunits can mediate the effects of GABA [31]. Spontaneous
and evoked Inhibitory PostSynaptic Potentials were reduced in amplitude but
never abolished by TPMPA, the antagonist of GABAC
receptors; however, they were completely blocked by bicuculline, the antagonist
of GABAA receptors [31]. The specific pharmacological properties of the
heteromeric complexes (composed of GABAA and
GABAC subunits) are similar to our data that bicuculline stopped GABA
from triggering sperm acrosome reaction, while TPMPA did not completely block
it [Fig. 4(B)] [16]. Thus, it is likely that GABAA and GABAC receptors are jointly involved in acrosome
reaction induced by GABA, similar to their roles in nervous system, although
this awaits further investigation.
In summary, our study demonstrates the presence of GABAC receptors in rat testis and spermatozoa. Our results suggest that
GABAC receptors are involved in rat sperm acrosome reaction. These results
add to the diversity of information on the neurotransmitter receptors in
mammalian spermatozoa.
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