BIPHASIC EFFECT OF GABA ON RAT SPERMACROSOME REACTION: INVOLVEMENTOF GABAA AND GABAB RECEPTORS

J.-H. HUX.-B. HEQ. WUY.-C. YAN

Laboratory of Molecular Cell Biology, Institute of Biochemistry andCell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, Shanghai,People’s Republic of China

S. S. KOIDE

Center for Biomedical Research, The Population Council,New York, USA

The functional relationship between GABAA and GABAB receptors in regulating acrosome reac-

tion (AR) of rat spermatozoa was demonstrated by studying the di� erential e� ects of a GABAB

agonist and an antagonist on the process. AR rates were determined using the chlortetracycline

staining assay. The induction of AR in rat sperm by GABA was found to be a biphasic phenom-

enon; i.e., AR rates increased with increasing GABA concentrations up to <5 mM and at higher

concentrations of the neurotransmitter (>5 mM), there was a reduction in the AR rates. This bipha-

sic phenomenon is apparently due to the di� erential interaction of the neurotransmitter with

GABA receptor subtypes in a dose-dependent manner; i.e., GABAA receptors (stimulatory) are pri-

marily activated at low concentration of GABA, while GABAB receptors (inhibitory) become ac-

tivated at higher concentrations. This hypothesis is supported by the present ®ndings that treatment

with saclofen, a GABAB receptor antagonist, did not in¯uence the AR rates e� ected by GABA at

low concentrations; while the AR rates were maintained at the maximum level at higher concentra-

tions of GABA, resulting in the elimination of the biphasic phenomenon. Baclofen, a GABAB re-

ceptor agonist, blocks the AR activating action of GABA at both low and high concentrations. It

would appear that the induction of AR in rat sperm by GABA is regulated by the proportionality

of activated GABAA and GABAB receptors acting as a yin±yang control.

Keywords baclofen, capacitation, chlortetracycline staining, fertilization, saclofen

This study was supported by a grant from the National ``973’’ project, No. G19990559.

Address correspondence to Dr. Yuan Chang Yan, PhD, Shanghai Institute of Biochemistry and Cell Biology,

Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, P. R. China.

E-mail: ycyan@sunm.shcnc.ac.cn

ARCHIVES OF ANDROLOGY 48:369±378 (2002)Copyright # 2002 Taylor & Francis0148-5016 /02 $12.00+.00DOI: 10.1080 /0148501029009924 6

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The GABAergic system has been well documented within the central nervous system(CNS) of vertebrates [18]. Peripheral tissues outside the CNS also possess this neuro-transmitter system [8]. In mammalian sperm, acrosome reaction (AR) is a modi®ed exo-cytotic event that is essential for successful fertilization [32]. AR will occur only withmammalian spermatozoa that have previously undergone the maturation process knownas capacitation. This maturation process requires a period of residence in the female repro-ductive tract or incubation in a speci®ed microenvironment [3, 16]. Two inducers of ARexist in oocyte vestments: the zona pellucida glycoprotein ZP3 and progesterone. Pro-gesterone induces AR in human sperm by interacting with a steroid receptor located in theplasma membrane. This membrane receptor is distinctly di� erent from the classical nu-clear receptor [4, 22, 26, 30] and may be related to the GABAA receptor=Cl7 channel [21].

In the CNS, GABAB receptors are coupled to potassium and calcium channels and tothe second messenger system by guanine nucleotide-binding (G) proteins [5]. Activation ofGABAB receptors has the long-lasting, modulatory e� ects of in¯uencing presynapticneurotransmission by decreasing Ca2‡ conductance and by increasing the postsynapticK ‡ conductance whereby generating late IPSPs [1, 29]. GABAB receptors are present inrat testis and sperm [14], although its function has not been clari®ed. Since rat spermcontain both the GABAA and GABAB receptors, the present study was performed todetermine the relationship of these subtypes of GABA receptors in regulating AR.

MATERIALS AND METHODS

Analytical-grade reagents were purchased from Shanghai Chemical Reagents (Shang-hai, P.R. China). Bovine serum albumin (BSA, fraction V), sodium pyruvate, sodiumlactate, GABA, progesterone, Hepes, saclofen, and baclofen were obtained from SigmaChemical (St. Louis, MO).

Male Wistar rats (3 months old) were decapitated and the caudal region of the epidi-dymides was freed of fat and blood vessels. Sperm were released from the caudal epidi-dymis and collected by the swim-up method [25]. To promote capacitation, theconcentration of spermatozoa was adjusted to a density of ¹1:0 £ 107=mL in a modi®edcapacitation medium (102 mM NaCl, 25 mM KCl, 1.7 mM CaCl2, 2.4 mM MgSO4,1.2 mM KH2PO4, 25 mM NaHCO3, 5 mM D-glucose, 5 mM sodium lactate, 0.25 mMsodium pyruvate, 20 mM Hepes, and 15 mg=mL BSA, pH 7.4) and incubated for 1.5 h at37¯C under an atmosphere of 5% CO2 [23].

For CTC staining to assess acrosome reaction, a modi®ed staining procedure of Fraserand Herod [10] was followed. CTC solution (1.5 mM) was prepared fresh with a bu� er(pH 7.8) containing 20 mM Tris, 130 mM NaCl, and 5 mM L-cysteine. The solution wasshielded from light at room temperature. After the addition of the agonist and=or an-tagonist, a 50-mL aliquot of the sperm suspension was mixed with an equal volume of CTCsolution, then 8 mL 12.5% (w=v) paraformaldehyde in PBS (pH 7.4) was added 30 s later.After gentle vortexing, 50 mL glycerol was introduced and mixed well with the spermsuspension to retard the fading of ¯uorescence. More than 250 cells were examined foreach sample analyzed.

Results are expressed as mean‹S.D. The data were analyzed with the Student’s t testfor comparison.

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RESULTS

E� ect of GABA on Capacitated Rat Sperm

Using the CTC staining assay, the status of the AR of rat sperm can be distinguished byexamining the patterns of ¯uorescent staining of the head region (Figure 1). Sperm withintact acrosome (Figure 1A) is characterized by a bright ¯uorescence over the entire head,while the acrosome-reacted sperm has a darkened head (no ¯uorescence) except for the tip(Figure 1C). Accordingly, any pattern between these 2 stages is classi®ed as the ``inter-mediate state’’ (Figure 1B) [10]. To determine the optimum concentration of GABA re-quired for the induction of AR, rat sperm were preincubated for 1.5 h in the capacitationmedium containing 1.5% BSA at 37¯C and under 5% CO2 in air, and were then challengedwith various concentrations of GABA (0.5±1000 mM, dissolved in 0.15 M NaCl) for15 min, or left untreated for an equal period of time. The AR rate of capacitated rat sperminduced with GABA varied with its concentration and was found to be a biphasic phe-nomenon (Figure 2). At GABA concentrations ranging from 0.5 to 5 mM, the percentageof sperm undergoing the AR increased, reaching the maximal rate at 5 mM. IncreasingGABA to higher concentrations (>5 mM) resulted in a decrease in the AR rates (Figure 2).The proportion of motile sperms in the control and treated specimens was equivalent (70±80%) at the end of the experiment.

E� ect of Saclofen on GABA-Initiated Rat Sperm AR

Capacitated rat sperms were treated with saclofen (200 mM, ®nal concentration), aGABAB receptor antagonist, for 10 min and AR induced with GABA at concentrationsranging from 0.5 to 1000 mM (Figure 3). Pretreatment with saclofen maintained the ARrates at the maxima even at high concentration of GABA, thereby eliminating the biphasicphenomenon (Figure 3).

Figure 1. CTC staining patterns of rat sperma at di� erent stages of AR. (A): Intact acrosome. Note

¯uorescence over the entire sperm head. (B) Intermediate state. Note darkened area below theequatorial segment. (C) Acrosome-reacted state. Note darkened area over the entire head except for

the tip. The staining procedure is described in the Methods section.

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Figure 2. E� ect of varying concentration of GABA in inducing acrosome reaction in rat sperm.

Values are means‹SD of 3 experiments. AR was determined by CTC staining.

Figure 3. E� ect of saclofen on rat sperm AR induced with varying concentrations of GABA. AR

was determined by CTC staining. Values are means‹SD of three experiments. *p<.05 (‹ saclofen,50 mM GABA); **p µ .01.

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E� ect of Baclofen on GABA-Initiated Rat Sperm AR

Capacitated rat spermatozoa were incubated with varying concentrations of baclofen(0±5 mM, in 0.15 M NaCl), a GABAB receptor agonist, for 10 min and AR induced with5 mM GABA (Figure 4). Baclofen at concentrations of 0.1±5 mM inhibited AR inducedwith 5 mM GABA in a dose-dependent manner (Figure 4A). Capacitated rat spermatozoapretreated with 50 mM baclofen failed to undergo AR upon treatment with GABA atvarying concentrations (0.5±1000 mM), (Figure 4B). Thus, activation of GABAB receptorsnulli®ed the stimulatory activity of GABAA receptors.

E� ect of Baclofen on Progesterone-Initiated Rat Sperm AR

Capacitated rat sperm were treated with 50 mM baclofen for 10 min and AR inducedwith 5 mM progesterone (dissolved in DMSO and diluted in 0.15 M NaCl) (Figure 5).Progesterone failed to trigger AR in capacitated rat spermatozoa pretreated with baclofen,demonstrating that activation of the GABAB receptors blocked the stimulatory action ofprogesterone mediated by GABAA receptors.

DISCUSSION

GABA, a major inhibitory neurotransmitte r in the mammalian CNS, also plays animportant physiological role in many systems outside the CNS [8, 18], including mam-malian fertilization. We have previously shown that GABAA and GABAB receptors areexpressed in rat testis and sperm [14, 15]. GABAB receptors are localized in the dorsalregion of the acrosome of rat spermatozoa [15], while the GABAA receptors are locatedprincipally on the ventral region of the acrosome (unpublished data). AR in rat sperma-tozoa is characterized by the disappearance of the ¯uorescent CTC staining of the head(Figure 1C), similar to the patterns observed with mouse sperm [31]. The present ®ndingssupport the hypothesis that GABA and progesterone trigger AR in rat sperm by activatingthe GABAA receptor pathway. This stimulatory capability of activated GABAA receptorscan be nulli®ed by the activation of GABAB receptors. The apparent function of GABAB

receptors is to maintain the acrosomes in a quiescent state and to prevent their prematureactivation. These regulatory actions of the subtypes of GABA receptors have beenclari®ed by studying the e� ects of GABAB receptor agonist baclofen and antagonistsaclofen.

The percentage of motile sperm before and after treatment with the drugs was the same.The optimal concentration of GABA in inducing AR in rat sperm is 5 mM. At lower(<5 mM) or higher concentrations (>5 mM), GABA is less e� ective. This biphasic phe-nomenon is similar to the observations made by Shi et al. [27, 28] with human and mousespermatozoa. The optimal concentration of GABA in initiating AR is ¹1.25 mM inhuman sperm and 0.5 mM in mouse sperm. Since the IC50 of GABA binding with nativerat brain GABAB receptors is 6.8‹0.36 mM [11], we hypothesized that GABA at highconcentration (>5 mM) may activate GABAB receptors, thereby nullifying the inducingactivity of the GABAA receptors. The net result is a decrease in the AR rate. This hy-pothesis is supported by the ®ndings that saclofen, a GABAB receptor antagonist,

GABA-Induced AR in Rat Sperm 373

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Figure 4. E� ect of baclofen on rat sperm AR induced with varying concentrations of GABA. ARwas determined by CTC staining. Values are means‹SD of 3 experiments. (A) E� ect of pre-

incubation with varying concentrations of baclofen on rat sperm. AR was induced with 5 mM

GABA. (B) E� ect of preincubation with 50 mM baclofen on rat sperm. AR was induced with varyingconcentrations of GABA. *p<.05; **p<.01.

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maintains the stimulatory activity of GABA at high concentrations (Figure 3) and ba-clofen, a GABAB receptor agonist, prevents the triggering of AR by GABA (Figure 4).

The biphasic e� ect of GABA on the induction of AR in rat spermatozoa is due to therelative proportion of activated GABAA and GABAB receptors in rat sperm. At lowerconcentrations (<5 mM), GABA activates primarily the GABAA receptors in a dose-dependent manner up to 5 mM. At higher concentrations (>5 mM), the AR rates fall dueto the activation of GABAB receptors. A similar situation occurs with progesterone-induced AR. The hormone appears to activate GABAA receptors, triggering AR in ratsperm. Treatment with baclofen, a GABAB receptor agonist, blocks the AR-inducingaction of the hormone. Thus, the induction of AR in rat sperm by progesterone ismediated by GABAA receptors.

The observed biphasic e� ect of GABA is not restricted to mammalian sperm. Thisphenomenon also occurs with other cell types. For example, the e� ect of GABA on thesecretion of the gonadotropin-releasing hormone (GnRH) by mouse GT1 neuronal cellsoccurs as a biphasic response [20]. Hence, within the CNS, GABA regulates neuronalactivity by interacting with both GABAA receptors and the G protein-coupled GABAB

receptors. In the mature nervous system, both receptor subtypes tend to reduce neuralexcitability; whereas in most neurons during development, activation of the GABAA re-ceptors tends to increase neural excitability by raising cytosolic Ca2‡ levels [6, 19];GABAB receptors play an inhibitory role in modulating the rise in Ca2‡ elicited by theactivation of GABAA receptors [24].

Figure 5. E� ect of baclofen on the GABA- and progesterone-induced AR in rat sperm. AR was

determined CTC staining. Values are means‹SD of 3 experiments. GABA, 5 mM; Bac (baclofen),

50 mM; P, progesterone, 5 mM. *p<.001.

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There may exist other mechanisms by which the biphasic e� ect of GABA in triggeringmammalian sperm AR is operational. In CNS, GABAB receptors are coupled to voltage-dependent calcium channels (VDCC) (N-, P-, and T- type) and reduce calcium current byblocking VDCC function [17]. However, activation of VDCC is essential for the occur-rence of AR in mammalian sperm initiated by ZP3, progesterone, and GABA [2, 9, 12, 27].We hypothesized that at low concentration, GABA interacts principally with GABAA

receptors in inducing AR via the VDCC-dependent pathway. At high concentrations,GABA activates the GABAB receptors coupled to G proteins, resulting in a blockage ofthe VDCC pathway. A second possible mechanism is based on studies showing that theactivation of GABAB receptors inhibits muscimol-stimulated uptake of 36Cl7 by themouse cerebellum. It is hypothesized that this action results from an activation of phos-pholipase C and the phosphorylation of GABAA receptors by protein kinase C [13]. Athird proposed mechanism is based on the ®ndings that the stimulatory response ofmelanotrophs of frog pituitary to GABAA receptors agonists is due to the production ofcAMP, while activation of GABAB receptors may block GABAA receptors function byinhibiting adenylate cyclase activity [7].

The present results strongly support the proposition that the relative proportion ofactivated GABAA and GABAB receptors at varying concentrations of GABA is the basisfor the biphasic phenomenon of AR observed with rat sperm. Activation of GABAA

receptors is stimulatory while that of GABAB receptors is inhibitory. The signal pathwayof how activation of GABAB receptors nullify the stimulatory action of GABAA receptorsneeds to be clari®ed.

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