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Identification and characterization of a novel splice variant of β3 subunit of GABAA receptor in rat testis and spermatozoa
The International Journal of Biochemistry & Cell Biology 37 (2005) 350–360
Identification and characterization of a novel splice variant of 3 subunit of GABAA receptor in rat testis and spermatozoa Shi-feng Lia,c,1 , Jing-hua Hua,1 , Yuan-chang Yana,b,∗ , Yu-guang Chenc , S.S. Koided , Yi-ping Lia,2 a
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China b Model Organism Division, E-Institutes of Shanghai Universities, PR China c College of Life Science, Shanghai University, Shanghai 200436, PR China d Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA Received 9 February 2004; received in revised form 22 June 2004; accepted 9 July 2004
Abstract ␥-Aminobutyric acid type A (GABAA ) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of 3 subunit of GABAA receptor, designated as 3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the 3 subunit of neuronal GABAA receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The 3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the 3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of 3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the 3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the 3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of 3 subunit of GABAA receptor, designated as 3t, is found in germ cells of rat testis and sperm. The inability of the 3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus. © 2004 Elsevier Ltd. All rights reserved. Keywords: GABAA receptor; 3 Subunit; Splice variant; Rat testis and spermatozoa
∗ 1 2
Corresponding author. Tel.: +86 21 54921395; fax: +86 21 64331090. E-mail address: [email protected] (Y.-c. Yan). Authors contributed equally to the work. Co-corresponding author.
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.07.008
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1. Introduction
2. Materials and Methods
␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and is found in many peripheral tissues (Akinci & Schofield, 1999). The receptors for GABA can be classified pharmacologically into three major subtypes, namely, ionotropic GABAA , GABAC and metabotropic GABAB receptors. GABAA receptor is the target of several centrally active drugs, including barbiturates and benzodiazepines (Burt & Kamatchi, 1991). Activation of the GABAA receptors results in the opening of chloride channels and polarization of the cell membrane. The GABAA receptor is a heteropentameric structure composed of various combinations of subunits belonging to at least three reported classes ␣,  and ␥ (Nayeem, Green, Martin, & Barnard, 1994). To date, six ␣ (␣1-␣6), four  (14), three ␥ (␥1-␥3), and one each of ␦, and subunit isoforms have been cloned from mammalian brain (Kittler et al., 2000). Brain GABAA receptors display high heterogeneity due to the existence of many different pentameric combination of at least 15 subunits (Stephenson, 1995). The mammalian sperm acrosome reaction (AR) is a modified exocytotic event that is essential to the fertilization process (Yanagimachi, 1994). Two main agonists of AR, namely the zona pellucida glycoprotein ZP3 (Kopf & Gerton, 1991) and progesterone (Meizel, Pillai, Diaz-Perez, & Thomas, 1990), have been identified in oocyte vestments. Recent studies suggest that GABAA or GABAA -like receptors may exist on mammalian spermatozoa. Progesterone appears to initiate AR by interacting with the neuronallike GABAA receptor/Cl− channel (Meizel, 1997; Shi & Roldan, 1995). In our previous study (Hu & Yan, 2002), GABAA receptors were detected in rat spermatozoa and found to be involved in AR triggered by GABA and by progesterone. The structure and composition of GABAA receptors of rat brain and testis and their function need to be determined. To achieve this goal, a full-length GABAA receptor 3 subunit of rat testis and sperm was isolated and identified. In addition, the cellular distribution of GABAA receptor 3 subunit in rat testis and brain and the subcellular localization in HEK 293 cells were determined.
2.1. Material
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The following chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, USA): bovine serum albumin (Fraction V), EDTA, mercaptoethanol, benzamidine, Triton X-100, Tween20, rabbit anti-goat IgG conjugated to horseradish peroxidase, rabbit anti-goat IgG conjugated to FITC, normal goat serum, DAB, Hochest 33258. Goat polyclonal antibody specific to the C-terminus of 3 subunit of GABAA receptor was obtained from Santa Cruz; Trizol from GIBCO; Oligotex kit from Qiagen and ECL reagent from Amersham Pharmacia Biotech. SMART RACE cDNA Amplification kit and Advantage 2 PCR kit were purchased from Clontech. T7/ SP6 DIG RNA Labeling Kit and DIG Nucleic Acid Detection Kit were from Roche; and ConA-Texas red from Molecular Probes. 2.2. Preparation of mRNA from rat testis and brain Rat testis and brain were freshly excised, and total RNA extracted using Trizol according to the procedure described by the manufacturer. mRNA was isolated using the Oligotex Kit. 2.3. RACE PCR 5 - and 3 -RACE were performed using the rat testis mRNA as template. The primers used in RACE PCR were designed by Primer 5 software as follows: 5 -RACE primer: 5 -CCCGGTTGATTTCACTCTTGGATCG-3 (corresponding to the neuronal GABAA receptor 3 subunit nucleotide sequence, 1127–1151). 3 -RACE primer: 5 -CATCCCGCACAAGAAGACGCACCTAC-3 (corresponding to the neuronal GABAA receptor 3 subunit nucleotide sequence, 1330–1355). The PCR reaction was performed with the PE GeneAmp Systems 9600, using the program described in the RACE user manual. The primary PCR product was diluted and subsequently used as the template in performing the secondary reaction.
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The RACE PCR products were TA-cloned into pGEM-T easy vector (Promega) and sequenced. 2.4. Northern blot analysis 3t mRNA was determined by Northern blot. Samples of isolated mRNA (2–5 g lane) were electrophoresed in 1% (w/v) formaldehyde agarose gel, capillary blotted onto a nylon membrane and crosslinked by exposure to UV. Probes (25 ng each) were random labeled with [␣-32 P]dCTP to a specific activity of >1 × 109 cpm. Hybridization for Northern blot was performed in 50% (v/v) formamide at 42 ◦ C. Membranes were washed in 0.2 × SSC, 0.1% (w/v) SDS at 55 ◦ C, and exposed to X-ray film with an intensifying screen, for 24 h or longer. 2.5. In situ hybridization In situ hybridization was performed using rat testis cryostat sections according to the method described by Komminoth (1992) and Long et al. (1992). Sense and antisense riboprobes were prepared with T7/SP6 DIG RNA Labeling Kit. Hybridization signal was detected by using DIG Nucleic Acid Detection Kit.
they were incubated with FITC-labeled rabbit anti-goat IgG for 1 h washed thrice to remove any unbound antibody. The stained spermatozoa were placed on a slide and examined by epifluorescence microscopy. 2.8. Construction of the GABAA receptor 3t-EGFPC chimera cDNA of the testis 3t subunit was produced using the polymerase chain reaction (PCR) with PstI and SacII sites primers (1: 5 CGC CTG CAG ATG GAG CGT CAT GTA GGC ATC 3 , 2: 5 CGC CCG CGG GTT AAC ATA GTA CAG CCA GTA 3 ). The cDNA of rat brain 3 subunit was produced at the BglII and PstI sites using primers (1: 5 CGC AGA TCT GAT GTG GGG CTT TGC GGG AGG 3 , 2: 5 CGC CTG CAG GTT AAC ATA GTA CAG CCA GTA AAC 3 ). The stop codon was replaced by restriction sites. The PCR products were subcloned into the pEGFP-N1 vector (Clontech) thereby fusing the EGFP gene to the COOH terminals of the 3t isoform and 3 subunit. The fidelity of the final expression constructions were verified by restrictive digestion and DNA sequencing. 2.9. Cell culture and transfection
2.6. Immunohistochemistry Immuohistochemical staining was perform using the indirect peroxidase-conjugated method with 10 m-thick frozen sections of rat testis. The cryosections were postfixed in 4% paraformaldehyde (w/v), air-dried, and stored at −20 ◦ C until used. Immunocytochemical staining was performed using anti-GABAA receptor 3 polyclonal antibody combined with rabbit anti-goat IgG HRP-conjugate and developed by incubating the slides in DAB substrate solution for 15–30 min. The reaction was stopped by washing the sections in distilled water. The slides were mounted and examined by light microscopy. 2.7. Immunocytofluorescence For indirect immunocytofluorescence localization of subunits, epididymal spermatozoa were fixed in methanol at −20 ◦ C for 30 min followed by 3% normal goat serum for 30 min to block nonspecific reaction, subsequently, the spermatozoa were incubated with primary antibody overnight at 4 ◦ C. After three washes,
Human embryonic kidney (HEK293) cells were maintained in a growth medium comprised of Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 25 g/ml penicillin and 100 g/ml streptomycin. Cells were incubated at 37 ◦ C under an atmosphere of 5% CO2 , 95% air. Cells were resuspended in phosphate buffered saline containing trypsin (25 g/ml) and EDTA (2 g/ml). The harvested cells were replated at 25–50% confluency in a 60 mm dish. Transfection was performed 20–24 h after subculturing with 10 g of plasmids (pEGFP-N1) containing the cDNAs for rat testis 3 and brain 3 GABAA receptor subunits by calcium phosphate precipitation. The transfected cells were washed and incubated for 24–48 h before further study. 2.10. Fluorescence and confocal microscopey The transfected cells were fixed in 4% paraformaldehyde for 30 min at 4 ◦ C and stained with Hochest 33258 (marker of nucleus) for 1 min at ambient temperature, washed with PBS and fixed with
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50% glycerol. The fixed cells were permeabilized with 0.1% Triton X-100 for 5 min in 0.1% trisodium citrate dihydrate solution and incubated with ConA-TexasRed (marker of ER membrane) for 1 h at ambient temperature, washed with PBS and fixed with 50% glycerol. The cells were examined using a laser scanning microscope (Bio-Rad) with a 60× oil immersion lens, 2.0×zoom, utilizing excitation at 488 nm for GFP, 543 nm for Texas Red and 330–385 nm for Hochest 33258.
tified by the method of RACE PCR. The full-length 3t consisted of 1937 bp (Fig. 1A) coding a polypeptide containing 471 amino acids. The 3t variant and 3 cDNA differed at the 5 -region of 480 bp consisting of 406 bp 5 -untranslated region and 74 bp 5 -open reading frame (ORF) region. The amino acid sequence of the unique segment of the deduced 3t polypeptide is shown in Fig. 1.
3. Results
Two probes were designed and used to localize 3t cDNA in rat testis. Probe-1 was amplified from the cDNA fragment obtained in our previous study (Hu, He Wu, Yan, & Koide, 2002). This fragment was found to be conserved in both subunits. Probe-2 was amplified from the 3t-specific 480 bp 5 -region cDNA. These two probes were labeled with [␣-32 P]dCTP or DIG,
3.1. Characterization of 3t cDNA and amino acids sequence of the deduced polypeptide The novel 3 subunit variant of GABAA receptor, designated as 3t, was isolated from rat testis and iden-
3.2. Existence and localization of 3t cDNA in rat testis
Fig. 1. Nucleotide sequence of 5 -terminal of 3t DNAs and amino-acid sequences of the N-terminals of the deduced polypeptides of rat testis 3t and brain 3 GABAA receptors. (A) nucleotide sequence of 5 terminal of 3t subunit cDNA. (***) start codon of 3t subunit; (· · ·) in-frame stop codon of 3t subunit in the 5 untranslated sequence; Arrow points to No. 151 nucleotide of rat brain 3 subunit. (B) Amino-acid sequence of the N-terminal of the deduced polypeptide of the novel 3t subunit variant of rat testis and brain 3 subunit. Except for the deduced 25 amino-acids in the N-terminal, the remainder of 3t is identical to that of rat brain 3. Arrow points to the potential signal peptide cleavage site of rat brain 3 subunit. ERHV underscored in the N-terminal of 3t is a potential endoplasmic reticulum retention signal sequence.
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Fig. 2. Northern blot of 3t subunit mRNA of GABAA receptor isolated from rat testis. The blot was hybridized with cDNA fragment (obtained from RT-PCR) radiolabeled by random priming. Te, rat testis poly(A)+ RNA; Br, rat brain poly(A)+ RNA. The blot was also probed with radiolabeled house-keeping GAPDH cDNA. (A) Probe-1; (B) Probe-2.
respectively, and used in the Northern blot (Fig. 2) and in the in situ hybridization assays (Fig. 3), respectively. Probe-1 recognized two transcripts, namely, a ∼6 and a ∼2.5 kb polyribonucleotides in the brain poly(A+ ) RNA sample and one transcript (∼6 kb) in testis poly(A+ ) RNA sample. Probe-2 recognized a single ∼6 kb transcript in the testis poly(A+ ) RNA sample; and none in brain sections. The size of the two transcripts isolated from the brain with Probe-1 is consistent with the reported size of neuronal 3 subunit transcripts (Ymer et al., 1989). 3 Subunit expression in rat brain was greater than that in rat testis. The in situ hybridization assays with DIG-labeled antisense Probes-1 and -2 (Fig. 3B and D) showed that the 3t transcript was localized in germ cells in the late step of spermatogenesis; whereas, no hybridization occurred, using DIG-labeled sense Probes-1 and -2 (Fig. 3A and C). 3.3. Immunolocalization of 3t in rat testis and spermatozoa The cellular localization of 3 antigen in rat testis and spermatozoa was validated using the immunohistochemical and immunocytofluorescence techniques. Positive immunoreactivity occurred principally in the region of the seminiferous tubules of rat testis sections containing elongated sperm (Fig. 4A and B). Immunocytofluorescence study with specific anti-GABAA re-
ceptor 3 polyclonal antibody showed that about 90% of the noncapacitated rat spermatozoa exhibited intense fluorescence mainly over the head of rat sperm and slight staining over the tail (Fig. 5). As control, no staining occurred when the primary antibody was replaced with normal goat serum or omitted (data not shown). 3.4. Expression and localization of GABAA receptor 3t splice variant in HEK293 cells The expression of the novel 3t variant and rat brain GABAA receptor 3 subunit were determined by confocal microscopy. 3t-EGFPC and 3-EGFPC chimeras were constructed by fusing the EGFP to its’ C-terminus region and transfected into HEK 293 cells respectively (Fig. 6). EGFP is uniformly distributed throughout the whole cell (Fig. 6A), same as that visualized by fluorescence microscopy (data dot shown). The expressed 3-EGFPC chimera product was detected in the cytoplasm and on the cell surface (Fig. 6B); whereas the 3t-EGFPC chimera product was retained within the cytoplasm. Two expression patterns were delineated (Fig. 6C and D). One pattern showed uniform distribution of fluorescence in the cytoplasm (Fig. 6C); whereas a second pattern consisted of intense green fluorescence mainly of some discrete intracellular compartments (Fig. 6D). To determine its subcellular site, the transfected cells were stained with ConA-Texas
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Fig. 3. In situ hybridization of GABAA receptor in the seminiferous tubules of rat testis. 3t Isoform transcript detected in cryostat sections of rat testis, using digoxigenin-labeled RNA probe. B: use of antisense Probe 1; D: application of antisense Probe 2; A and C; sense Probe1 and sense Probe 2 were used, respectively. Note intense staining of germ cells in the late stages of spermatogenesis, indicated by arrows (B and D). No signals were elicited with the use of sense probes (A and C).
Fig. 4. Immunohistochemical localization of 3 subunit of GABAA receptor in rat testis. Sections were stained with specific polyclonal antiGABAA receptor 3 antibody (A) followed by overlaying with the corresponding HRP-labeled second antibody and counterstained. Staining did not occur when the primary antibody was replaced with non-immune goat serum (B). Arrows point to a layer of positive stained cells consisting of spermatozoa.
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Fig. 5. Indirect immunofluorescence localization of C-terminus antigen of 3 GABAA receptor on rat spermatozoa. Paraformaldehyde-fixed, noncapacitated rat spermatozoa were incubated with the specific polyclonal anti-3 GABAA receptor antibody followed by overlaying with corresponding FITC-labeled second antibody. Note intense staining of the sperm head and slight staining of the tail (A). Observation with phase contrast microscope (B).
Red (Fig. 7). The green and the red fluorescences were localized in the same compartment, indicating that the expression products of the novel 3t variant was confined to the cytoplasm, perhaps retained in the endoplasmic reticulum.
4. Discussion Recent reported studies demonstrate the presence of a GABAergic system in mammalian testis and sperm, including GABAA receptor, GABAB receptor and GABA transporter (GAT1) (Ma et al., 2000; Hu, He, & Yan, 2000; He, Hu, Wu, Yan, & Koide, 2001; Hu et al., 2002; He et al., 2001; Geigerseder et al., 2003). In the present study, the 3 -RACE PCR product was found to be identical to the neuronal 3 subunit. However, the 5 -RACE PCR product differs from neuronal 3 subunit in the 5 -region. By using two different probes, 3t transcript was found to be expressed in rat spermatocytes (Figs. 2 and 3) but not in the brain. The expression of 3t transcript, however, is low compared to 3 expression in the brain. The structural variation in the 5 regions of 3t and 3 transcripts suggests the possibility that their formation, may be controlled by different mechanisms. Differential regulatory mechanisms may
account for the finding that a single ∼6 kb transcript was detected in the testis poly(A+ ) RNA. Antibody specific to the C-terminus of neuronal 3 subunit, structurally similar to the deduced 3t protein, has been used to determine the presence of 3t isoform in rat germ cells and spermatozoa. Immunohistochemical and immunocytofluorescent stainings show that the 3t antigen is located on the sperm head, confirming the results of a previous study (Hu et al., 2002). The deduced unique polypeptide segment (25 amino acids) in the N-terminus of the 3t isoform differs from that of the 3 subunit. Since this sequence in rat brain 3 subunit has the characteristic of a cleavable signal peptide, the corresponding segment of the 3t variant might act as a signal peptide. In spite of containing similar basic residues and several hydrophobic residues (Fig. 1), characteristic of a signal peptide, analysis with PSORT II software did not confer this property. Moreover, the results suggest that the ERHV region in the 25 amino acid segment might be an endoplasmic reticulum (ER) retention signal. The GABAA receptor-GFP chimera has been successfully used to study the subcelluar localization and assembly of GABAA receptor subunits (Bueno, Robinson, Alvarez-Hernandez, & Leidenheimer, 1998; Kittler et al., 2000). When 3tEGFPC chimera is expressed in HEK293 cell, two different patterns of expression occurs (Figs. 6 and 7).
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Fig. 6. Distribution of 3t-EGFPC chimera in HEK 293 cells: (A) cells were transfected with pEGFP-N1 vector. Note bright green fluorescence distributed throughout the entire cell; (B) the control product of 3-EGFPC chimeras is detected either on the cell membrane and within the cytoplasm. The arrows point to the area of cell membrane; (C) product of 3t-EGFPC chimera failed to reach the plasma membrane and is evenly distributed in the cytoplasm; (D) alternate 3t-EGFPC chimera expression pattern, note bright green fluorescence located predominantly at discrete intracellular sites. All images were obtained by confocal microscopy (Bio-rad, Radiance 2100). (bar = 10 m).
In both patterns, they were found that the 3t isoform cannot reach the cell membrane. This is different to rat brain 3 subunit which targets the cell surface and forms functional Cl− channels when expressed homomerically in a variety of cell types (Davies, Kirkness, & Hales, 1997). These results indicate that the GABAA receptor 2 subunit may be an essential factor for the translocation of ␣1-EGFP to the surface plasmalemma (Connor, Boileau, & Czajkowski, 1998). The cell surface targeting of ␥2L-GFPN is dependent on the co-
expression with the receptor 2 and ␣1 subunits (Kittler et al., 2000). It is possible that the cell surface expression of 3t-EGFPC may be dependent upon the existence of some cell factors or coexpression with other GABAA receptor subunits. To determine the intracellular distribution of the novel variant in HEK 293 cells, the transfected cells were treated with ConA-Texas-Red, a marker for the ER membrane (Shnyder & Hubbard, 2002). The red fluorescence of ConA-Texas-Red and the green fluores-
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Fig. 7. Subcelluar localization of 3t-EGFPC chimera in HEK 293 cells. Cells were permeabilized and stained with ConA-Texas Red. A, one 3t-EGFPC chimera expression pattern B. alternate 3t-EGFPC chimera expression pattern. The green fluorescence and the red fluorescence localized in the same region. The arrows point to the sites (yellow) of colocalization. All images were obtained by confocal microscopy (Bio-rad, Radiance 2100). (bar = 10 m).
cence of 3t-EGFPC chimera are colocalized (Fig. 7), indicating that the novel 3 variant may be retained in the ER. Furthermore, ConA can also bind to the Golgi complex (Guasch, Guerri, & O’Connor, 1993). Thus the novel 3t variant may be incorporated into the Golgi apparatus. It is conceivable that the 3t variant is synthesized in the ER and transported to the Golgi apparatus. Moreover, the lack of a signal peptide may cause the 3t variant to be retained in the cytoplasm and prevents its access to the cell surface. During the spermatogenesis, the development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the spermatozoon. During this phase, several cellular organelles (nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, centriole, etc.) simultaneously undergo structural and biochemical changes that require a series of shape changes from round to elongate as spermatid metamorphoses into a developing spermatozoon. In this study, the results of in situ hybridization indicated that the transcript of this variant may occur since the secondary sper-
matocyte and possibly through to elongated spermatid (Goodwin, Karabinus, & Pergolizzi, 2000; Paradisi et al., 2001). This variant targets to the sperm membrane finally with the existence of other subunits. Molecular cloning has revealed the presence of different variants and splicing of 3 subunit in rat testis. Regulation of RNA splicing confers upon genes a modality that adds flexibility. Tissue-specific mRNA splicing events are prevalent in natural proteins, such as integrin, adrenocorticosteroid receptors, oestrogen receptor (Sonnenberg, 1993; Herman, 1993; Blankenstein et al., 1995). It is noteworthy that IGF gene expression is influenced by the nature of the transcription start site by effecting the length and the sequence of the IGF mRNA 5 -UTR, which can subsequently regulate the translatability of mRNA and the production of the coded protein and potentially, the intracellular processing and secretion of the final gene product (LeRoith & Roberts, 1993). Similarly, the occurrence of mRNA splicing is also observed with GABAA receptor ␥2 subunit and GABAB receptor (Zhang, Ashiya, Sherman, & Grabowski, 1996; Isomoto et al., 1998). Nonetheless,
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details about these important molecular mechanisms of transcriptional gene control need to be elucidated. In conclusion, evidences obtained in the present study show that the occurrence of alternative mRNA splicing and the existence of a single variant designated as 3t isoform of GABAA receptor in rat spermatocytes and spermatozoa. The present results contribute information toward a better understanding of the molecular basis of a testis-specific splicing event. Simultaneously, the data clarify the transport and target pathway of GABAA receptor in rat testis and sperm. Further studies need to be performed to determine the cellular role of 3t transcript, its function within the cytoplasm, and the final subcellular destination of the 3t isoform. Acknowledgements The present study were supported by a grant from “973” program supported by the Ministry of National Science and Technology, No. G19990559 and in part by E-institutes of Shanghai Municipal Education Commission. References Akinci, M. K., & Schofield, P. R. (1999). Widespread expression of GABAA receptor subunits in peripheral tissues. Neuroscience Research, 35, 145–153. Blankenstein, M. A., Koehorst, S. G., van der Kallen, C. J., Jacobs, H. M., van Spriel, A. B., Donker, G. H., et al. (1995). Oestrogen receptor independent expression of progestin receptors in human meningioma–a review. Journal of Steroid Biochemical Molecular Biology, 53, 361–365. Bueno, O. F., Robinson, L. C., Alvarez-Hernandez, X., & Leidenheimer, N. J. (1998). Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes. Molecular Brain Research, 59, 165–177. Burt, D. R., & Kamatchi, G. L. (1991). GABAA receptor subtypes: from pharmacology to molecular biology. FASEB Journal, 5, 2916–2923. Connor, J. X., Boileau, A. J., & Czajkowski, C. (1998). A GABAA receptor ␣1 subunit tagged with green fluorescent protein requires a  subunit for functional surface expression. Journal of Biological Chemistry, 273, 28906–28911. Davies, P. A., Kirkness, E. F., & Hales, T. G. (1997). Modulation by general anaesthetics of rat GABAA receptors comprised of ␣13 and 3 subunits expressed in human embryonic kidney 293 cells. British Pharmacology, 120, 899–909. Geigerseder, C., Doepner, R., Thalhammer, A., Frungieri, M. A., Gamel-Didelon, K., Calandra, R. S., et al. (2003). Evidence for
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Identification and characterization of a novel splice variant of 3 subunit of GABAA receptor in rat testis and spermatozoa Shi-feng Lia,c,1 , Jing-hua Hua,1 , Yuan-chang Yana,b,∗ , Yu-guang Chenc , S.S. Koided , Yi-ping Lia,2 a
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China b Model Organism Division, E-Institutes of Shanghai Universities, PR China c College of Life Science, Shanghai University, Shanghai 200436, PR China d Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA Received 9 February 2004; received in revised form 22 June 2004; accepted 9 July 2004
Abstract ␥-Aminobutyric acid type A (GABAA ) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of 3 subunit of GABAA receptor, designated as 3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the 3 subunit of neuronal GABAA receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The 3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the 3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of 3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the 3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the 3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of 3 subunit of GABAA receptor, designated as 3t, is found in germ cells of rat testis and sperm. The inability of the 3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus. © 2004 Elsevier Ltd. All rights reserved. Keywords: GABAA receptor; 3 Subunit; Splice variant; Rat testis and spermatozoa
∗ 1 2
Corresponding author. Tel.: +86 21 54921395; fax: +86 21 64331090. E-mail address: [email protected] (Y.-c. Yan). Authors contributed equally to the work. Co-corresponding author.
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.07.008
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1. Introduction
2. Materials and Methods
␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and is found in many peripheral tissues (Akinci & Schofield, 1999). The receptors for GABA can be classified pharmacologically into three major subtypes, namely, ionotropic GABAA , GABAC and metabotropic GABAB receptors. GABAA receptor is the target of several centrally active drugs, including barbiturates and benzodiazepines (Burt & Kamatchi, 1991). Activation of the GABAA receptors results in the opening of chloride channels and polarization of the cell membrane. The GABAA receptor is a heteropentameric structure composed of various combinations of subunits belonging to at least three reported classes ␣,  and ␥ (Nayeem, Green, Martin, & Barnard, 1994). To date, six ␣ (␣1-␣6), four  (14), three ␥ (␥1-␥3), and one each of ␦, and subunit isoforms have been cloned from mammalian brain (Kittler et al., 2000). Brain GABAA receptors display high heterogeneity due to the existence of many different pentameric combination of at least 15 subunits (Stephenson, 1995). The mammalian sperm acrosome reaction (AR) is a modified exocytotic event that is essential to the fertilization process (Yanagimachi, 1994). Two main agonists of AR, namely the zona pellucida glycoprotein ZP3 (Kopf & Gerton, 1991) and progesterone (Meizel, Pillai, Diaz-Perez, & Thomas, 1990), have been identified in oocyte vestments. Recent studies suggest that GABAA or GABAA -like receptors may exist on mammalian spermatozoa. Progesterone appears to initiate AR by interacting with the neuronallike GABAA receptor/Cl− channel (Meizel, 1997; Shi & Roldan, 1995). In our previous study (Hu & Yan, 2002), GABAA receptors were detected in rat spermatozoa and found to be involved in AR triggered by GABA and by progesterone. The structure and composition of GABAA receptors of rat brain and testis and their function need to be determined. To achieve this goal, a full-length GABAA receptor 3 subunit of rat testis and sperm was isolated and identified. In addition, the cellular distribution of GABAA receptor 3 subunit in rat testis and brain and the subcellular localization in HEK 293 cells were determined.
2.1. Material
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The following chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, USA): bovine serum albumin (Fraction V), EDTA, mercaptoethanol, benzamidine, Triton X-100, Tween20, rabbit anti-goat IgG conjugated to horseradish peroxidase, rabbit anti-goat IgG conjugated to FITC, normal goat serum, DAB, Hochest 33258. Goat polyclonal antibody specific to the C-terminus of 3 subunit of GABAA receptor was obtained from Santa Cruz; Trizol from GIBCO; Oligotex kit from Qiagen and ECL reagent from Amersham Pharmacia Biotech. SMART RACE cDNA Amplification kit and Advantage 2 PCR kit were purchased from Clontech. T7/ SP6 DIG RNA Labeling Kit and DIG Nucleic Acid Detection Kit were from Roche; and ConA-Texas red from Molecular Probes. 2.2. Preparation of mRNA from rat testis and brain Rat testis and brain were freshly excised, and total RNA extracted using Trizol according to the procedure described by the manufacturer. mRNA was isolated using the Oligotex Kit. 2.3. RACE PCR 5 - and 3 -RACE were performed using the rat testis mRNA as template. The primers used in RACE PCR were designed by Primer 5 software as follows: 5 -RACE primer: 5 -CCCGGTTGATTTCACTCTTGGATCG-3 (corresponding to the neuronal GABAA receptor 3 subunit nucleotide sequence, 1127–1151). 3 -RACE primer: 5 -CATCCCGCACAAGAAGACGCACCTAC-3 (corresponding to the neuronal GABAA receptor 3 subunit nucleotide sequence, 1330–1355). The PCR reaction was performed with the PE GeneAmp Systems 9600, using the program described in the RACE user manual. The primary PCR product was diluted and subsequently used as the template in performing the secondary reaction.
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The RACE PCR products were TA-cloned into pGEM-T easy vector (Promega) and sequenced. 2.4. Northern blot analysis 3t mRNA was determined by Northern blot. Samples of isolated mRNA (2–5 g lane) were electrophoresed in 1% (w/v) formaldehyde agarose gel, capillary blotted onto a nylon membrane and crosslinked by exposure to UV. Probes (25 ng each) were random labeled with [␣-32 P]dCTP to a specific activity of >1 × 109 cpm. Hybridization for Northern blot was performed in 50% (v/v) formamide at 42 ◦ C. Membranes were washed in 0.2 × SSC, 0.1% (w/v) SDS at 55 ◦ C, and exposed to X-ray film with an intensifying screen, for 24 h or longer. 2.5. In situ hybridization In situ hybridization was performed using rat testis cryostat sections according to the method described by Komminoth (1992) and Long et al. (1992). Sense and antisense riboprobes were prepared with T7/SP6 DIG RNA Labeling Kit. Hybridization signal was detected by using DIG Nucleic Acid Detection Kit.
they were incubated with FITC-labeled rabbit anti-goat IgG for 1 h washed thrice to remove any unbound antibody. The stained spermatozoa were placed on a slide and examined by epifluorescence microscopy. 2.8. Construction of the GABAA receptor 3t-EGFPC chimera cDNA of the testis 3t subunit was produced using the polymerase chain reaction (PCR) with PstI and SacII sites primers (1: 5 CGC CTG CAG ATG GAG CGT CAT GTA GGC ATC 3 , 2: 5 CGC CCG CGG GTT AAC ATA GTA CAG CCA GTA 3 ). The cDNA of rat brain 3 subunit was produced at the BglII and PstI sites using primers (1: 5 CGC AGA TCT GAT GTG GGG CTT TGC GGG AGG 3 , 2: 5 CGC CTG CAG GTT AAC ATA GTA CAG CCA GTA AAC 3 ). The stop codon was replaced by restriction sites. The PCR products were subcloned into the pEGFP-N1 vector (Clontech) thereby fusing the EGFP gene to the COOH terminals of the 3t isoform and 3 subunit. The fidelity of the final expression constructions were verified by restrictive digestion and DNA sequencing. 2.9. Cell culture and transfection
2.6. Immunohistochemistry Immuohistochemical staining was perform using the indirect peroxidase-conjugated method with 10 m-thick frozen sections of rat testis. The cryosections were postfixed in 4% paraformaldehyde (w/v), air-dried, and stored at −20 ◦ C until used. Immunocytochemical staining was performed using anti-GABAA receptor 3 polyclonal antibody combined with rabbit anti-goat IgG HRP-conjugate and developed by incubating the slides in DAB substrate solution for 15–30 min. The reaction was stopped by washing the sections in distilled water. The slides were mounted and examined by light microscopy. 2.7. Immunocytofluorescence For indirect immunocytofluorescence localization of subunits, epididymal spermatozoa were fixed in methanol at −20 ◦ C for 30 min followed by 3% normal goat serum for 30 min to block nonspecific reaction, subsequently, the spermatozoa were incubated with primary antibody overnight at 4 ◦ C. After three washes,
Human embryonic kidney (HEK293) cells were maintained in a growth medium comprised of Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 25 g/ml penicillin and 100 g/ml streptomycin. Cells were incubated at 37 ◦ C under an atmosphere of 5% CO2 , 95% air. Cells were resuspended in phosphate buffered saline containing trypsin (25 g/ml) and EDTA (2 g/ml). The harvested cells were replated at 25–50% confluency in a 60 mm dish. Transfection was performed 20–24 h after subculturing with 10 g of plasmids (pEGFP-N1) containing the cDNAs for rat testis 3 and brain 3 GABAA receptor subunits by calcium phosphate precipitation. The transfected cells were washed and incubated for 24–48 h before further study. 2.10. Fluorescence and confocal microscopey The transfected cells were fixed in 4% paraformaldehyde for 30 min at 4 ◦ C and stained with Hochest 33258 (marker of nucleus) for 1 min at ambient temperature, washed with PBS and fixed with
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50% glycerol. The fixed cells were permeabilized with 0.1% Triton X-100 for 5 min in 0.1% trisodium citrate dihydrate solution and incubated with ConA-TexasRed (marker of ER membrane) for 1 h at ambient temperature, washed with PBS and fixed with 50% glycerol. The cells were examined using a laser scanning microscope (Bio-Rad) with a 60× oil immersion lens, 2.0×zoom, utilizing excitation at 488 nm for GFP, 543 nm for Texas Red and 330–385 nm for Hochest 33258.
tified by the method of RACE PCR. The full-length 3t consisted of 1937 bp (Fig. 1A) coding a polypeptide containing 471 amino acids. The 3t variant and 3 cDNA differed at the 5 -region of 480 bp consisting of 406 bp 5 -untranslated region and 74 bp 5 -open reading frame (ORF) region. The amino acid sequence of the unique segment of the deduced 3t polypeptide is shown in Fig. 1.
3. Results
Two probes were designed and used to localize 3t cDNA in rat testis. Probe-1 was amplified from the cDNA fragment obtained in our previous study (Hu, He Wu, Yan, & Koide, 2002). This fragment was found to be conserved in both subunits. Probe-2 was amplified from the 3t-specific 480 bp 5 -region cDNA. These two probes were labeled with [␣-32 P]dCTP or DIG,
3.1. Characterization of 3t cDNA and amino acids sequence of the deduced polypeptide The novel 3 subunit variant of GABAA receptor, designated as 3t, was isolated from rat testis and iden-
3.2. Existence and localization of 3t cDNA in rat testis
Fig. 1. Nucleotide sequence of 5 -terminal of 3t DNAs and amino-acid sequences of the N-terminals of the deduced polypeptides of rat testis 3t and brain 3 GABAA receptors. (A) nucleotide sequence of 5 terminal of 3t subunit cDNA. (***) start codon of 3t subunit; (· · ·) in-frame stop codon of 3t subunit in the 5 untranslated sequence; Arrow points to No. 151 nucleotide of rat brain 3 subunit. (B) Amino-acid sequence of the N-terminal of the deduced polypeptide of the novel 3t subunit variant of rat testis and brain 3 subunit. Except for the deduced 25 amino-acids in the N-terminal, the remainder of 3t is identical to that of rat brain 3. Arrow points to the potential signal peptide cleavage site of rat brain 3 subunit. ERHV underscored in the N-terminal of 3t is a potential endoplasmic reticulum retention signal sequence.
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Fig. 2. Northern blot of 3t subunit mRNA of GABAA receptor isolated from rat testis. The blot was hybridized with cDNA fragment (obtained from RT-PCR) radiolabeled by random priming. Te, rat testis poly(A)+ RNA; Br, rat brain poly(A)+ RNA. The blot was also probed with radiolabeled house-keeping GAPDH cDNA. (A) Probe-1; (B) Probe-2.
respectively, and used in the Northern blot (Fig. 2) and in the in situ hybridization assays (Fig. 3), respectively. Probe-1 recognized two transcripts, namely, a ∼6 and a ∼2.5 kb polyribonucleotides in the brain poly(A+ ) RNA sample and one transcript (∼6 kb) in testis poly(A+ ) RNA sample. Probe-2 recognized a single ∼6 kb transcript in the testis poly(A+ ) RNA sample; and none in brain sections. The size of the two transcripts isolated from the brain with Probe-1 is consistent with the reported size of neuronal 3 subunit transcripts (Ymer et al., 1989). 3 Subunit expression in rat brain was greater than that in rat testis. The in situ hybridization assays with DIG-labeled antisense Probes-1 and -2 (Fig. 3B and D) showed that the 3t transcript was localized in germ cells in the late step of spermatogenesis; whereas, no hybridization occurred, using DIG-labeled sense Probes-1 and -2 (Fig. 3A and C). 3.3. Immunolocalization of 3t in rat testis and spermatozoa The cellular localization of 3 antigen in rat testis and spermatozoa was validated using the immunohistochemical and immunocytofluorescence techniques. Positive immunoreactivity occurred principally in the region of the seminiferous tubules of rat testis sections containing elongated sperm (Fig. 4A and B). Immunocytofluorescence study with specific anti-GABAA re-
ceptor 3 polyclonal antibody showed that about 90% of the noncapacitated rat spermatozoa exhibited intense fluorescence mainly over the head of rat sperm and slight staining over the tail (Fig. 5). As control, no staining occurred when the primary antibody was replaced with normal goat serum or omitted (data not shown). 3.4. Expression and localization of GABAA receptor 3t splice variant in HEK293 cells The expression of the novel 3t variant and rat brain GABAA receptor 3 subunit were determined by confocal microscopy. 3t-EGFPC and 3-EGFPC chimeras were constructed by fusing the EGFP to its’ C-terminus region and transfected into HEK 293 cells respectively (Fig. 6). EGFP is uniformly distributed throughout the whole cell (Fig. 6A), same as that visualized by fluorescence microscopy (data dot shown). The expressed 3-EGFPC chimera product was detected in the cytoplasm and on the cell surface (Fig. 6B); whereas the 3t-EGFPC chimera product was retained within the cytoplasm. Two expression patterns were delineated (Fig. 6C and D). One pattern showed uniform distribution of fluorescence in the cytoplasm (Fig. 6C); whereas a second pattern consisted of intense green fluorescence mainly of some discrete intracellular compartments (Fig. 6D). To determine its subcellular site, the transfected cells were stained with ConA-Texas
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Fig. 3. In situ hybridization of GABAA receptor in the seminiferous tubules of rat testis. 3t Isoform transcript detected in cryostat sections of rat testis, using digoxigenin-labeled RNA probe. B: use of antisense Probe 1; D: application of antisense Probe 2; A and C; sense Probe1 and sense Probe 2 were used, respectively. Note intense staining of germ cells in the late stages of spermatogenesis, indicated by arrows (B and D). No signals were elicited with the use of sense probes (A and C).
Fig. 4. Immunohistochemical localization of 3 subunit of GABAA receptor in rat testis. Sections were stained with specific polyclonal antiGABAA receptor 3 antibody (A) followed by overlaying with the corresponding HRP-labeled second antibody and counterstained. Staining did not occur when the primary antibody was replaced with non-immune goat serum (B). Arrows point to a layer of positive stained cells consisting of spermatozoa.
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Fig. 5. Indirect immunofluorescence localization of C-terminus antigen of 3 GABAA receptor on rat spermatozoa. Paraformaldehyde-fixed, noncapacitated rat spermatozoa were incubated with the specific polyclonal anti-3 GABAA receptor antibody followed by overlaying with corresponding FITC-labeled second antibody. Note intense staining of the sperm head and slight staining of the tail (A). Observation with phase contrast microscope (B).
Red (Fig. 7). The green and the red fluorescences were localized in the same compartment, indicating that the expression products of the novel 3t variant was confined to the cytoplasm, perhaps retained in the endoplasmic reticulum.
4. Discussion Recent reported studies demonstrate the presence of a GABAergic system in mammalian testis and sperm, including GABAA receptor, GABAB receptor and GABA transporter (GAT1) (Ma et al., 2000; Hu, He, & Yan, 2000; He, Hu, Wu, Yan, & Koide, 2001; Hu et al., 2002; He et al., 2001; Geigerseder et al., 2003). In the present study, the 3 -RACE PCR product was found to be identical to the neuronal 3 subunit. However, the 5 -RACE PCR product differs from neuronal 3 subunit in the 5 -region. By using two different probes, 3t transcript was found to be expressed in rat spermatocytes (Figs. 2 and 3) but not in the brain. The expression of 3t transcript, however, is low compared to 3 expression in the brain. The structural variation in the 5 regions of 3t and 3 transcripts suggests the possibility that their formation, may be controlled by different mechanisms. Differential regulatory mechanisms may
account for the finding that a single ∼6 kb transcript was detected in the testis poly(A+ ) RNA. Antibody specific to the C-terminus of neuronal 3 subunit, structurally similar to the deduced 3t protein, has been used to determine the presence of 3t isoform in rat germ cells and spermatozoa. Immunohistochemical and immunocytofluorescent stainings show that the 3t antigen is located on the sperm head, confirming the results of a previous study (Hu et al., 2002). The deduced unique polypeptide segment (25 amino acids) in the N-terminus of the 3t isoform differs from that of the 3 subunit. Since this sequence in rat brain 3 subunit has the characteristic of a cleavable signal peptide, the corresponding segment of the 3t variant might act as a signal peptide. In spite of containing similar basic residues and several hydrophobic residues (Fig. 1), characteristic of a signal peptide, analysis with PSORT II software did not confer this property. Moreover, the results suggest that the ERHV region in the 25 amino acid segment might be an endoplasmic reticulum (ER) retention signal. The GABAA receptor-GFP chimera has been successfully used to study the subcelluar localization and assembly of GABAA receptor subunits (Bueno, Robinson, Alvarez-Hernandez, & Leidenheimer, 1998; Kittler et al., 2000). When 3tEGFPC chimera is expressed in HEK293 cell, two different patterns of expression occurs (Figs. 6 and 7).
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Fig. 6. Distribution of 3t-EGFPC chimera in HEK 293 cells: (A) cells were transfected with pEGFP-N1 vector. Note bright green fluorescence distributed throughout the entire cell; (B) the control product of 3-EGFPC chimeras is detected either on the cell membrane and within the cytoplasm. The arrows point to the area of cell membrane; (C) product of 3t-EGFPC chimera failed to reach the plasma membrane and is evenly distributed in the cytoplasm; (D) alternate 3t-EGFPC chimera expression pattern, note bright green fluorescence located predominantly at discrete intracellular sites. All images were obtained by confocal microscopy (Bio-rad, Radiance 2100). (bar = 10 m).
In both patterns, they were found that the 3t isoform cannot reach the cell membrane. This is different to rat brain 3 subunit which targets the cell surface and forms functional Cl− channels when expressed homomerically in a variety of cell types (Davies, Kirkness, & Hales, 1997). These results indicate that the GABAA receptor 2 subunit may be an essential factor for the translocation of ␣1-EGFP to the surface plasmalemma (Connor, Boileau, & Czajkowski, 1998). The cell surface targeting of ␥2L-GFPN is dependent on the co-
expression with the receptor 2 and ␣1 subunits (Kittler et al., 2000). It is possible that the cell surface expression of 3t-EGFPC may be dependent upon the existence of some cell factors or coexpression with other GABAA receptor subunits. To determine the intracellular distribution of the novel variant in HEK 293 cells, the transfected cells were treated with ConA-Texas-Red, a marker for the ER membrane (Shnyder & Hubbard, 2002). The red fluorescence of ConA-Texas-Red and the green fluores-
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Fig. 7. Subcelluar localization of 3t-EGFPC chimera in HEK 293 cells. Cells were permeabilized and stained with ConA-Texas Red. A, one 3t-EGFPC chimera expression pattern B. alternate 3t-EGFPC chimera expression pattern. The green fluorescence and the red fluorescence localized in the same region. The arrows point to the sites (yellow) of colocalization. All images were obtained by confocal microscopy (Bio-rad, Radiance 2100). (bar = 10 m).
cence of 3t-EGFPC chimera are colocalized (Fig. 7), indicating that the novel 3 variant may be retained in the ER. Furthermore, ConA can also bind to the Golgi complex (Guasch, Guerri, & O’Connor, 1993). Thus the novel 3t variant may be incorporated into the Golgi apparatus. It is conceivable that the 3t variant is synthesized in the ER and transported to the Golgi apparatus. Moreover, the lack of a signal peptide may cause the 3t variant to be retained in the cytoplasm and prevents its access to the cell surface. During the spermatogenesis, the development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the spermatozoon. During this phase, several cellular organelles (nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, centriole, etc.) simultaneously undergo structural and biochemical changes that require a series of shape changes from round to elongate as spermatid metamorphoses into a developing spermatozoon. In this study, the results of in situ hybridization indicated that the transcript of this variant may occur since the secondary sper-
matocyte and possibly through to elongated spermatid (Goodwin, Karabinus, & Pergolizzi, 2000; Paradisi et al., 2001). This variant targets to the sperm membrane finally with the existence of other subunits. Molecular cloning has revealed the presence of different variants and splicing of 3 subunit in rat testis. Regulation of RNA splicing confers upon genes a modality that adds flexibility. Tissue-specific mRNA splicing events are prevalent in natural proteins, such as integrin, adrenocorticosteroid receptors, oestrogen receptor (Sonnenberg, 1993; Herman, 1993; Blankenstein et al., 1995). It is noteworthy that IGF gene expression is influenced by the nature of the transcription start site by effecting the length and the sequence of the IGF mRNA 5 -UTR, which can subsequently regulate the translatability of mRNA and the production of the coded protein and potentially, the intracellular processing and secretion of the final gene product (LeRoith & Roberts, 1993). Similarly, the occurrence of mRNA splicing is also observed with GABAA receptor ␥2 subunit and GABAB receptor (Zhang, Ashiya, Sherman, & Grabowski, 1996; Isomoto et al., 1998). Nonetheless,
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details about these important molecular mechanisms of transcriptional gene control need to be elucidated. In conclusion, evidences obtained in the present study show that the occurrence of alternative mRNA splicing and the existence of a single variant designated as 3t isoform of GABAA receptor in rat spermatocytes and spermatozoa. The present results contribute information toward a better understanding of the molecular basis of a testis-specific splicing event. Simultaneously, the data clarify the transport and target pathway of GABAA receptor in rat testis and sperm. Further studies need to be performed to determine the cellular role of 3t transcript, its function within the cytoplasm, and the final subcellular destination of the 3t isoform. Acknowledgements The present study were supported by a grant from “973” program supported by the Ministry of National Science and Technology, No. G19990559 and in part by E-institutes of Shanghai Municipal Education Commission. References Akinci, M. K., & Schofield, P. R. (1999). Widespread expression of GABAA receptor subunits in peripheral tissues. Neuroscience Research, 35, 145–153. Blankenstein, M. A., Koehorst, S. G., van der Kallen, C. J., Jacobs, H. M., van Spriel, A. B., Donker, G. H., et al. (1995). Oestrogen receptor independent expression of progestin receptors in human meningioma–a review. Journal of Steroid Biochemical Molecular Biology, 53, 361–365. Bueno, O. F., Robinson, L. C., Alvarez-Hernandez, X., & Leidenheimer, N. J. (1998). Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes. Molecular Brain Research, 59, 165–177. Burt, D. R., & Kamatchi, G. L. (1991). GABAA receptor subtypes: from pharmacology to molecular biology. FASEB Journal, 5, 2916–2923. Connor, J. X., Boileau, A. J., & Czajkowski, C. (1998). A GABAA receptor ␣1 subunit tagged with green fluorescent protein requires a  subunit for functional surface expression. Journal of Biological Chemistry, 273, 28906–28911. Davies, P. A., Kirkness, E. F., & Hales, T. G. (1997). Modulation by general anaesthetics of rat GABAA receptors comprised of ␣13 and 3 subunits expressed in human embryonic kidney 293 cells. British Pharmacology, 120, 899–909. Geigerseder, C., Doepner, R., Thalhammer, A., Frungieri, M. A., Gamel-Didelon, K., Calandra, R. S., et al. (2003). Evidence for
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