The isoform type of g subunits of GABAA receptor is a molecular determinant of its pharmacological characteristics. At present, the existence of GABAA receptor in mammalian sperm is still a controversy. By using degenerate primers designed according to highly conserved region in all three γ (γ1, γ2 and γ3) subunits cloned in rat brain, we performed reverse transcription polymerase chain reaction (RT-PCR) to examine the expression pattern of g subunits of GABAA receptor in rat testis. Only one 370 bp fragment was obtained from RT-PCR in rat testis and sequencing results showed that it represented g1 subunit, but not γ2 or γ3 subunit. Using the cloned \fragment as probe, a 3.8 kb transcript which in size as same as γ1 subunit in rat brain was detected in rat testis mRNA by performing Northern blot assay. Furthermore, results of in situ hybridization assay confirmed that γ1 subunit was expressed in round spermatids and spermatozoa, maybe also in secondary spermatocyte. These evidences proved that γ1 subunit of GABAA receptor is exclusively expressed in rat testis and this feature may be the structural basis of the specific function of GABAA receptors in sperm acrosome reaction.
γ-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the vertebrate central nervous system (CNS)1. Whereas outside the CNS, many peripheral tissues have also been found to have GABAergic system2.
The mammalian sperm acrosome reaction (AR) is a modified exocytotic event that is essential to the fertilization process3. Two main agonists of AR, the zona pellucida glycoprotein ZP34 and progesterone5, have been identified in the oocyte vestments. Recent studies have suggested that progesterone appears to initiate the human AR by acting at a novel type of steroid receptor on the sperm plasma membrane but not by the classical nuclear receptor6, 7, 8, 9, 10. Meantime, a great deal of researches so far suggested the participation of a neuronal-like GABAA recepor/Cl− channel in progesterone-initiated mammalian sperm AR11, 12. Moreover, it is also found that GABA at relatively low concentrations may mimic the effects of progesterone: increasing the fraction of acrosome-reacted cells in capacitated human and mouse spermatozoa10, 13. However, it is a conspicuous lack about the molecular characteristics of this receptor on mammalian spermatozoa. Given the promotion in understanding the functional mechanism of progesterone on mammalian spermatozoa, it is significant to identify the subunits composition of sperm GABAA receptor.
All natural GABAA receptors, found in vivo so far, containing α, β and γ subunits, meanwhile, unique pharmacological profiles could be conferred by GABAA receptors containing different g subunits 14. In previous work (unpublished), we have found α5, β1, β2, and β3 subunits of GABAA receptor were expressed in rat testis and finally localized onto sperm head. Thus, we designed experiments to examine which type of γ subunits exists in rat testis. And the identification of expression type of γ subunits in testis might provide significant structural information for the understanding about the specific effects of GABAA receptors on sperm AR.
MATERIALS AND METHODS
Preparation of mRNA from rat testis
Rat (Wistar male, 90 days of age) testis was freshly excised. Testis total RNA was extracted using Trizol (Gibco-BRL) according to the procedure described by the manufacturer. mRNA was isolated using Oligotex Kit (Qiagen).
Rat testis first strand cDNA was synthesized using Superscript Preamplification System (Gibco-BRL).
Two degenerate PCR primers were designed based on the conserved domain among three members of GABAA receptor g subunits as
(corresponding to g1 cDNA sequence: 574-592);
(corresponding to g1 cDNA sequence: 924-942).
Primers were designed to encompass the intronic sequences, so that any PCR product amplified from genomic DNA contaminating the RNA preparation could be distinguished. The PCR reaction was performed initially by denaturation at 95°C for 4 min, and then 30 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min followed by extension for 8 min at 72°C. The primary PCR product was diluted and then used as template to perform a secondary reaction and the reaction condition is as same as the first round except for the annealing temperature increasing to 55°C
PCR products were TA-cloned into PGEMT easy vector (Promega) and sequenced.
Northern blot analysis
The existence of γ1 mRNA was examined by performing Northern blotting. Isolated poly(A)+ RNA (1-3 mg each lane) was electrophoresed in a 1% (w/v) formaldehyde agarose gel, capillary blotted onto a nylon membrane and UV cross-linked. Probes (25 ng each) were random labeled with [32P]dCTP to a specific activity of > 1× 109 cpm. Blots were hybridized in a standard 50%(v/v) formamide buffer15 at 42 °C and washed in 0.2 × SSC, 0.1 (w/v) %SDS at 55°C before exposure to X-ray film with an intensifying screen. Autoradiographs were exposed for 24 h or longer.
In situ hybridization
In situ hybridization of rat testis cryostat sections was performed as Long A.A. described16. Sense and antisense riboprobes were prepared with T7/ SP6 DIG RNA Labeling Kit (Roche). Hybridization signal was detected by using DIG Nucleic Acid Detection Kit (Roche).
As shown in Fig 1, after performing RT-PCR with degenerate primers, an intense ∼370 bp band which corresponding in size to the fragments of predicted brain γ subunits was obtained from testis mRNA specimen. Cloning and sequencing this fragment, results showed that only the transcript of γ1 subunit fragment, but not that of γ2 or γ3 subunit fragment, exist in rat testis mRNA. The sequence of cloned γ1 subunit fragment in testis is exact identical to that in brain (seq: 574-942).
Northern blot analysis
Using the fragment obtained from RT-PCR as probe and labeled by [32P]dCTP, we performed Northern blot analysis. As shown in Fig 2A, rat testis mRNA contains one hybridizing bands at ∼ 3.8 kb which is identical in size to the γ1 band detected in rat brain mRNA. It is obvious that much lower level of γ1 expression was found in testis compared with that in brain. Result of hybridizing to house-keeping gene GAPDH with same blot was shown in Fig 2B.
In situ hybridization
As shown in Fig 3A, C and D, results of in situ hybridization with DIG-labeled antisense γ1 probe showed that γ1 transcript localizes in round spermatid and spermatozoa, maybe also in secondary spermatocyte, region of rat testis section, but not in spermatogonia, Leydig cells and Sertoli cells; while with DIG-labeled sense γ1 probe didn't show the positive signal, as shown in Fig 3B.
Our evidences directly showed that the transcript of γ1 subunit of GABAA receptor exists exclusively in rat testis. Based on the results of Northern blotting, it is obvious that the level of γ1 subunit in rat testis is much lower than that in rat brain, and this is in consistent with the finding that g1 subunit band was obtained from testis cDNA after second PCR amplification. Meanwhile, results of in situ hybridization localized the expression of γ1 subunit in round spermatids and spermatozoa, maybe also in secondary spermatocyte, but not in other cell types in rat testis.
Progesterone or GABA can induce mammalian sperm acrosome reaction in vitro; and previous studies have presumed that, after stimulated by progesterone or GABA, the depolarization of sperm membrane and calcium influx induced by the activation of GABAA receptors on spermatozoa can lead to the sperm acrosome reaction11. These “excitatory” effects induced by activation of GABAA receptors on spermatozoa opposite to the classical inhibitory effects of GABAA receptors in mature mammalian CNS17. However, it is not clear about the mechanism responsible for these effects yet. Electrophysiological studies showed that GABAA receptors with different subunits composition have different physiological function; moreover, evidences in vitro suggested unique pharmacological profiles could be conferred by GABAA receptors containing different gamma subunits14. For example, it is suggested that the positive modulatory effect of DMCM, one modulator of GABAA receptor, depends on the presence of g1 subunit in the native GABAA receptors18; moreover, studies on recombinant GABAA receptors showed that the presence of γ1 subunit induced higher neurosteroid modulatory efficacy on GABAA receptors compared with the presence of γ2 subunit19. In contrast to γ2 subunit being abundantly expressed in mature mammalian CNS20,γ1subunit is exclusively expressed in rat testis. Thus, we speculated that γ1 subunit in rat testis might contribute to the specific function of GABAA receptors on the spermatozoa. Interestingly, other studies also suggested a possible relationship between γ1 subunit and excitatory function of GABAA receptors in a few cases. It is accepted that during early development stage GABA exerts mainly an excitatory action via GABAA receptors through membrane depolarization and a rise in intracellular Ca2+, and these effects disappeared during development21, 22, while studies on distribution of subunits of GABAA receptor revealed that g1 subunit was strongly expressed during early development stage and with development its expression dropped markedly and restricted to relatively few structures23, whereas γ2 subunit becomes the mainly expression type of gamma subunit in mature CNS20. Additionally, the excitatory effects of GABAA receptors were also proved in bromodeoxyundine (BrdU)+ neural precursor cells and γ1 is also the expression pattern of gamma subunit in these cells24. In conclusion, we hypothesized that γ1 subunit might be a molecular determinant of the excitatory function of GABAA receptor in rat spermatozoa.
Our evidences proved directly that γ1 subunit is the only type of gamma subunits expressed in rat testis and thus confirmed the expression of GABAA receptor in rat testis. This study provided the significant structural information for making investigation into the “excitatory” function mechanism of GABAA receptors in mammalian fertilization process. Moreover, due to previous studies having demonstrated directly that GABAB receptor25 and GAT-126, 27 are also present on spermatozoa, so further studies will also be carried out to examine the possible relationship between GABAA receptor, GABAB receptor and GAT-1 in sperm AR.
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This work was supported by the grant from National Key Basic Research Project, “973” No. G199905592.
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