|
|
|
Original Paper
|
|
|||
|
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 ρ 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 manufacturer�s 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'-AACGAGCGGTTCCGATGCC�CTGAG-3' and reverse primer 5'-TGTCGCCTTCAC�CG�T��TC�CAGTT-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 Student�s 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 [16,17,21�23]. 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.
References
1�� Chebib M, Johnston GA.
GABA-activated ligand-gated ion channels: medicinal chemistry and molecular
biology. J Med Chem 2000, 43: 1427-1447
2�� Bormann J. The �ABC� of
GABA receptors. Trends Pharmacol Sci 2000, 21: 16-19
3�� Enz R, Cutting GR.
Molecular composition of GABAC receptors. Vision Res
1998, 38: 1431-1441
4�� Chebib M. GABAC receptor ion
channels. Clin Exp Pharmacol Physiol 2004, 31: 800-804
5�� Enz R, Brandstatter JH,
Hartveit E, Wassle H, Bormann J. Expression of GABA receptor r1 and r2 subunits in the
retina and brain of the rat. Eur J Neurosci 1995, 7: 1495-1501
6�� Rozzo A, Armellin M,
Franzot J, Chiaruttini C, Nistri A, Tongiorgi E. Expression and dendritic mRNA
localization of GABAC receptor r1 and r2 subunits in
developing rat brain and spinal cord. Eur J Neurosci 2002, 15: 1747-1758
7�� Boue-Grabot E, Taupignon
A, Tramu G, Garret M. Molecular and electrophysiological evidence for a GABAC receptor in
thyrotropin-secreting cells. Endocrinology 2000, 141: 1627-1632
8�� Jansen A, Hoepfner M,
Herzig KH, Riecken EO, Scherubl H. GABA(C) receptors in
neuroendocrine gut cells: a new GABA-binding site in the gut. Pflugers
Arch 2000, 441: 294-300
9�� Gamel-Didelon K, Kunz L,
Fohr KJ, Gratzl M, Mayerhofer A. Molecular and physiological evidence for
functional gamma-aminobutyric acid (GABA)-C receptors in growth
hormone-secreting cells. J Biol Chem 2003, 278: 20192-20195
10� Meizel S. The sperm, a neuron
with a tail: neuronal receptors in mammalian sperm. Biol Rev Camb Philos
Soc 2004, 79: 713-732
11� Erdo SL, Wolff JR. g-Aminobutyric acid
outside the mammalian brain. J Neurochem 1990, 54: 363-372
12� Akinci MK, Schofield PR.
Widespread expression of GABA(A) receptor subunits in
peripheral tissues. Neurosci Res 1999, 35: 145-153
13� Shi QX, Yuan YY, Roldan ER. g-Aminobutyric acid
(GABA) induces the acrosome reaction in human spermatozoa. Mol Hum
Reprod 1997, 3: 677-683
14� Roldan ER, Murase T, Shi QX.
Exocytosis in spermatozoa in response to progesterone and zona pellucida.
Science 1994, 266: 1578-1581
15� Erdo SL, Rosdy B, Szporny L.
Higher GABA concentrations in fallopian tube than in brain of the rat. J
Neurochem 1982, 38: 1174-1176
16� Hu JH, He XB, Wu Q, Yan YC,
Koide SS. Subunit composition and function of GABAA receptors of rat
spermatozoa. Neurochem Res 2002, 27: 195-199
17� He XB, Hu JH, Wu Q, Yan YC,
Koide SS. Identification of GABA(B) receptor in rat testis and
sperm. Biochem Biophys Res Commun 2001, 283: 243-247
18� Hu JH, He XB, Wu Q, Yan YC,
Koide SS. Biphasic effect of GABA on rat sperm acrosome reaction: Involvement
of GABA(A) and GABA(B) receptors.
Arch Androl 2002, 48: 369-378
19� Herr JC, Thomas D, Bush LA,
Coonrod S, Khole V, Howards SS, Flickinger CJ. Sperm mitochondria-associated
cysteine-rich protein (SMCP) is an autoantigen in Lewis rats. Biol
Reprod 1999, 61: 428-435
20 Ragozzino D, Woodward RM, Murata
Y, Eusebi F, Overman LE, Miledi R. Design and in vitro pharmacology of a
selective g-aminobutyric acid C receptor antagonist. Mol
Pharmacol 1996, 50: 1024-1030
21� Hu JH, He XB, Yan YC.
Identification of gamma-aminobutyric acid transporter (GAT1) on the rat sperm.
Cell Res 2000, 10: 51-58
22� Hu JH, Yan YC. Identification
of gamma1 subunit of GABA(A) receptor in rat testis.
Cell Res 2002, 12: 33-37
23� Li SF, Hu JH, Yan YC, Chen YG,
Koide SS, Li YP. Identification and characterization of a novel splice variant
of b3 subunit of GABA(A) receptor in rat testis and
spermatozoa. Int J Biochem Cell Biol 2005, 37: 350-360
24� Geigerseder C, Doepner RF,
Thalhammer A, Krieger A, Mayerhofer A. Stimulation of TM3 Leydig cell
proliferation via GABA(A) receptors: a new role for
testicular GABA. Reprod Biol Endocrinol 2004, 2: 13
25� Turner RM. Moving to the beat:
a review of mammalian sperm motility regulation. Reprod Fertil Dev 2006,
18: 25-38
26� Calogero AE, Hall J, Fishel S,
Green S, Hunter A, D�Agata R. Effects of g-aminobutyric acid
on human sperm motility and hyperactivation. Mol Hum Reprod 1996, 2: 733-738
27� Ritta MN, Calamera JC, Bas DE.
Occurrence of GABA and GABA receptors in human spermatozoa. Mol Hum
Reprod 1998, 4: 769-773
28� Shi QX, Roldan ER. Evidence
that a GABAA-like receptor is involved in progesterone-induced
acrosomal exocytosis in mouse spermatozoa. Biol Reprod 1995, 52: 373-381
29� Fletcher EL, Koulen P, Wassle
H. GABAA and GABAC receptors on mammalian rod
bipolar cells. J Comp Neurol 1998, 396: 351-365
30� Akasu T, Munakata Y, Tsurusaki
M, Hasuo H. Role of GABAA and GABAC receptors in the
biphasic GABA responses in neurons of the rat major pelvic ganglia. J
Neurophysiol 1999, 82: 1489-1496
31� Milligan CJ, Buckley NJ,
Garret M, Deuchars J, Deuchars SA. Evidence for inhibition mediated by
coassembly of GABAA and GABAC receptor subunits in native
central neurons. J Neurosci 2004, 24: 7241-7250