Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 67, 46-54 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

A BKCa to Kv Switch During Spermatogenesis in the Rat Seminiferous Tubules1

X.D. Gonga, J.C.H. Lia, G.P.H. Leunga, K.H. Cheunga, and P.Y.D. Wong2,a

a Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a complex cellular event during which the diploid germ cells differentiate and divide by mitosis and meiosis at specific time points along the spermatogenic cycle to generate the haploid spermatozoa. For this complex event to go in an orderly manner, cell differentiation and division must be precisely controlled by signals arising from within and outside the seminiferous tubules. Changes in the membrane potential of the germ cells are likely to be an important part of the signaling mechanism. We have applied the whole-cell patch clamp technique to identify and characterize ion channels in different spermatogenic cells from immature and mature rat testes fractionated by discontinuous Percoll gradient. A voltage- and Ca2+- dependent, outwardly rectifying current with gating and pharmacologic properties resembling the large conductance K+ channels (BKCa) was recorded from the spermatogonia and primary spermatocytes. Another voltage-dependent, outwardly rectifying current that was sensitive to 4-aminopyridine, a Kv channel blocker, was detected in spermatocytes and early spermatids. This current is likely caused by the smaller conductance, voltage-sensitive K+ channels (Kv). In some spermatogonia, both the BKCa channels and the Kv channels could be simultaneously detected in the same cell. It appears that during the course of spermatogenesis, there is up-regulation of Kv but down-regulation of BKCa. Reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry further confirmed the differential expression of the ion channels in different spermatogenic cells. We conclude that these ion channels may play an important role in the control of spermatogenesis.

gamete biology, sperm, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a complex cellular event during which germ cells in the seminiferous tubules proliferate and divide by mitosis and then meiosis to generate the haploid gametes, the spermatozoa [1, 2]. During the process, the developing germ cells migrate from the basal to the adluminal side of the epithelium. Each spermatogonium divides by mitosis into a clone of spermatogonia. Some of the spermatogonia are destined to produce the gametes and differentiate into primary spermatocytes. When they do so they migrate through the tight junctions into the adluminal compartment, inside which each primary spermatocyte undergoes meiosis to produce 2 haploid secondary spermatocytes. Each secondary spermatocyte then divides and forms 2 round spermatids. From this stage onward, there is no more cell division, and the round spermatids differentiate into the final spermatozoa by a process called spermiogenesis, during which substantial remodeling of germ cell occurs. Several morphologic and biochemical changes in male germ cells were reported to be associated with spermatogenesis [1, 3, 4].

Spermatogenesis entails cell division and differentiation at specific time points along the spermatogenic cycle. For this complex event to go in an orderly fashion, tight and precise control of the steps by signals arising from within and outside of the seminiferous tubules is expected. As with many physiologic processes, changes in membrane potentials of the germ cells are likely to be an important part of the signaling mechanisms [5, 6]. It is known that certain ion channel blockers and altered ionic conditions can inhibit sperm motility, sperm maturation, and the acrosome reaction [7]. Ion channel mechanisms have been investigated in germ cells and spermatozoa using voltage- and ion-sensitive dyes, bilayer reconstitution, DNA recombinant techniques, cDNA expression in heterologous systems, immunocytochemistry, pharmacology, and, to lesser extent, patch clamping [8]. Several ion channels such as an inward rectifier K+ channel [9], cyclic nucleotide-gated channels [10], and pH-sensitive K+ channels [11] have been described in the testis, where they may have an important role in spermatogenesis and reproduction. Recently, a voltage-gated cation channel thought to play a role in sperm motility has been identified in the tail of mouse sperm [12, 13].

In the present study, we have applied the whole-cell patch clamp technique to study ion channels in dissociated rat spermatogenic cells. We report that a large conductance, voltage- and Ca2+-dependent potassium channel is active in spermatogonia but is rarely observed in cells in later stages of spermatogenesis, such as spermatocytes and spermatids. Another voltage-dependent potassium channel, seemingly a Kv1.3 channel, is present in spermatocytes and spermatids but not in spermatogonia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Rat Germ Cells

All experiments on animals were performed in accordance with the guidelines on the use of laboratory animals laid down by the Animal Ethics Committee of the Chinese University of Hong Kong. Male Sprague-Dawley rats 80 ± 10 days old (~300 g body weight) were used as a source of the spermatocytes and spermatids. Male rats 15 ± 5 days old (~20 g body weight) were used as a source of the spermatogonia and primary spermatocytes. Total germ cells were isolated by a mechanical procedure without the use of any enzymatic treatments, as previously described [14]. Briefly, rats were killed by CO2 inhalation. The testes were dissected out and immersed in sterile PBS (140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4). The testes were decapsulated, and the blood vessels were removed. Dispersed seminiferous tubules were washed twice by sedimentation in gravity to remove Leydig cells and red blood cells. Seminiferous tubules were then minced in a Petri dish with sterile blades and scalpels for 10 min. The minced tubules were washed 3 times with PBS at 200 x g for 2 min each. The supernatants containing germ cells were pooled and filtered consecutively through mira cloth, 100-µm nylon mesh, glass wool, and 20-µm nylon mesh to remove tissue debris, cell clumps, elongated spermatids, and spermatozoa. The filtrate was washed 4 times with PBS at 800 x g for 4 min each. Cells were then reconstituted in serum-free Ham F12 nutrient mixture (F12) and Dulbecco modified Eagle medium (DMEM) (1:1, v/v; F12-DMEM), supplemented with 6 mM sodium lactate, 2 mM sodium pyruvate, 20 mg/l gentamicin, and 10 µg/ml bacitracin. Somatic cells such as Sertoli cells, Leydig cells, and fibroblasts were removed by incubating the cell suspension in a humidified atmosphere of 95% air and 5% CO2 (v/v) at 32°C for 2–3 h. Those loosely adherent cells were recovered and washed twice with PBS at 200 x g for 2 min each. When the final preparation was analyzed for somatic cell contamination by various criteria as detailed elsewhere [14, 15], a negligible amount of somatic cell contamination was noted. Germ cells with viability greater than 95% as demonstrated by trypan blue dye exclusion test were used in the isolation of different spermatogenic cells, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis.

Purification of Spermatogenic Cells by Discontinuous Percoll Gradient

To purify spermatogenic cells from total germ cells, a discontinuous Percoll gradient of 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 60% was prepared in F12-DMEM with modifications [16]. A total of 10–30 x 106 germ cells obtained from the 15 ± 5-day-old rats (largely spermatogonia and primary spermatocytes) and the 80 ± 10-day-old rats (largely spermatocytes and spermatids) were suspended in 500 µl of F12-DMEM. These cell suspensions were layered carefully on top of the 20% Percoll gradients in two 15-ml centrifuge tubes. Centrifugation was carried out at 1164 x g for 15 min at 20°C. After centrifugation, the positions of the major bands containing highly purified germ cells were noted and recorded. In general, cellular debris was found in 20%–25%, spermatocytes in 30%–35%, spermatids in 35%–40%, and spermatogonia in 45% of Percoll layers. Cells in each layers were pooled accordingly, resuspended in 10 ml of F12-DMEM, pelleted by centrifugation at 280 x g for 10 min, and washed twice to remove residual Percoll. The cell viability was greater than 95% as shown by trypan blue dye exclusion test. Approximately 2 x 105 cells were gently reconstituted in 2 ml of serum-free F12-DMEM supplemented with 6 mM sodium lactate, 2 mM sodium pyruvate, 20 mg/l gentamicin, and 10 µg/ml bacitracin, and plated into poly-D-lysine coated 35-mm dishes for patch clamp recording.

Analysis of germ cell populations by DNA flow cytometry was performed essentially as previously described [14, 17]. Briefly, for assessing the immature germ cell population, germ cells isolated from 15-day-old rats by the mechanical procedure [14] were treated with pepsin (0.5% [w/v] pepsin in PBS buffer, pH 2.0), ethanol fixed, and stained with PBS containing 125 mg/ml ethidium bromide, 0.005% RNase A (w/v), and 0.3% Nonidet P-40 (v/v). At least 10 000 cells were acquired for each sample in a FACScan Flow Cytometer (Becton Dickinson, San Jose, CA).

For 15-day-old rats, DNA flow cytometry revealed that the relative percentage of spermatogonia (2N) and primary spermatocytes (4N) was 47% and 51%, respectively. This result is consistent with that of an earlier study in which the cell population was analyzed by microscopy [18]. For adult rats, DNA flow cytometry reveals that the cell preparation consists largely of spermatogonia, spermatocytes, and round spermatids with relative percentages of 17%, 18%, and 65%, respectively [14, 15].

Reverse Transcription-Polymerase Chain Reaction

The presence of BKCa and Kv mRNA in rat germ cells was assessed by RT-PCR. Total RNA was isolated from dissociated rat germ cells using TRIzol reagent (Gibco BRL, Gaithersburg, MD). A 2-µg sample of total RNA was used for first-strand cDNA synthesis using oligo(dT)18 primer and Superscript II RNase H- Reverse Transcriptase (SuperScript Preamplification System; Gibco BRL). The resulting first-strand cDNA was directly used for PCR amplification.

Different sets of primers were designed and synthesized for PCR analysis. The primer pair for BKCa were as follows: sense 5'-AGATGGATGCGCTCATCATC-3' and antisense 5'-TTCACTTCCAGCCAGAACCA-3' [19], which specifically amplify the coding region from 543 to 1090 and yield a PCR product of 547 base pair (bp). The primer pair for amplifying Kv1.3 was as follows: sense 5'-CATTCTAAGGGGCT-GCAGAT-3' and antisense 5'-CCTTGATGAATGGTCTGGAA-3' [20], which specifically amplify the coding region from 987 to 1678 and yield a PCR product of 691 bp. We used S-16 as an internal standard and coamplified it in the PCR reaction. The two primers used for amplifying S-16 were as follows: 5'-TCCCGTGCAGTCCGTTCAAGTCTT-3' and antisense 5'-GCCAAACTTCTTGGATTCGCAGCG-3' [21], which yield a PCR product of 385 bp. Reactions were carried out under the following conditions: denaturation at 94°C for 30 sec, annealing at 53°C for 45 sec, and extension at 72°C for 1 min. A total of 26 cycles were performed. The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by staining with ethidium bromide. To confirm the authenticity of these amplicons, PCR products of expected sizes were gel-purified and subjected to partial DNA sequencing (Applied Biosystems 310 genetic analyzer; Applied Biosystems, Foster City, CA).

Western Blot Analysis

To assess BKCa and Kv proteins in germ cell membranes, germ cell cytosols were prepared from their corresponding primary cultures with minor modifications [22]. Germ cells were briefly rinsed with lysis buffer (20 mM Tris, pH 7.4 containing 1 mM EDTA, 2 mM PMSF). Thereafter, cells were resuspended in lysis buffer at 3 times their packed cell volume and incubated at 4°C for 1 h. The cell sample was then centrifuged at 15 000 x g for 30 min at 4°C, and the supernatant was used as cytosol. Cell pellets were resuspended in solubilization buffer (lysis buffer containing 0.1% SDS [w/v] and 0.1% Triton X-100 [v/v]), incubated at 4°C for 1 h, and centrifuged at 40 000 x g for 90 min at 4°C. The supernatant collected was designated membrane extract. The protein content was estimated by the Coomassie blue dye-binding assay with BSA as a standard [23]. Approximately 75 µg of cytosol and membrane extracts were resolved on a 7% T polyacrylamide gel under denaturing and reducing conditions as described previously [24]. The immunoreactive BKCa and Kv1.3 bands were visualized by staining the corresponding blot with the anti-BKCa and anti-Kv1.3 antibodies (Alomone Labs, Jerusalem, Israel).

Immunohistochemistry

To localize Kv and BKCa channels within mature testes, paraffin sections (3 µm) of mature rat testes were dewaxed and hydrated. Antigens were retrieved by treatment in 0.01 M citrate buffer (pH 6.0) for 5 min in a microwave oven. The sections were then rinsed twice with pure water and incubated in methanol containing 3% H2O2 for 15 min. After a rinse with pure water and PBS, sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector PK-6101; Vector Labs, Burlingame, CA) for 30 min and then with the polyclonal anti-Kv1.3 antibody or anti-BKCa antibody (Alomone Labs), diluted 1:100 with diluting buffer (PBS with 0.01% Triton X-100, 0.01% Tween 20, and 0.1% BSA) at 4°C overnight. Sections were washed 3 times with PBS and incubated with biotinylated secondary antibody (ABC kit) for 30 min. After 3 washes with PBS, the sections were incubated with Vectastain Elite ABC reagent (ABC kit) for 30 min and finally washed three times with PBS again. Visualization was achieved by immersing sections in a peroxidase substrate solution (Vector VIP substrate kit) until the desired stain intensity developed. Slides were rinsed with pure water for 5 min, counterstained with Lillie-Mayer hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted for observation. Negative controls were obtained by omission of primary antibodies.

Whole-Cell Patch Clamp Recordings

Whole-cell patch clamp recordings were made to study ion channels in dissociated rat spermatogenic cells. After an approximately 30-min incubation at 32°C, germ cells adhered to the bottom of the dish. Recordings were made from solitary cells that settled on the bottom of the dish and had smooth margins. Recordings were performed at room temperature (20–25°C) using an Axopatch 200B amplifier and DigiData 1200 series Interface (Axon Instruments, Foster City, CA). Patch pipettes (2–5 M{Omega}) were pulled from 1.0-mm outer diameter, 0.5-mm inner diameter borosilicate glass pipettes (Sutter Instrument Co., Novato, CA) using a horizontal puller (Sutter). They were polished before use. Ionic current was recorded using conventional whole-cell patch clamp technique. The cell membrane potential was held at -70 mV. Signals were filtered at 1 kHz, then digitized with DigiData 1200 (Axon Instruments). The sampling rate was set at 100 µsec. The pClamp (version 8.0) program was used for data recording and analysis. Normally, the bath contained Krebs Henseleit solution (117 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24.8 mM NaHCO3, 11.1 mM glucose, pH adjusted to 7.4 with 0.3 N NaOH). The pipette solution varied according to the experiments but normally contained 140 mM KCl. In experiments in which single-channel current was recorded, distribution of the amplitude of channel currents was constructed using the Fetchan program (version 8.0; Axon Instruments). Inhibition of whole-cell current by pharmacologic agents was expressed as a percentage of the control current. Values were expressed as mean ± SEM The Student t-test was used to compare different groups of data, and P < 0.05 was considered statistically significant.

Chemicals

Fetal bovine serum, DMEM, and nonessential amino acids were purchased from Gibco Laboratories (Grand Island, NY). Tetraethylammonium (TEA), 4-aminopyridine (4-AP), gentamicin, sodium lactate, sodium pyruvate, bacitracin, poly-D-lysine, and penicillin/streptomycin were from Sigma (St. Louis, MO). Tetrodotoxin was purchased from Tocris Cookson Ltd. (London, U.K.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Measurement of Membrane Potentials of Germ Cells

The perforated-patch method was used for measurement of spermatogenic cell membrane potentials. The internal solution contained 140 mM KCl and nystatin at a concentration of 100 µg/ml. To increase the chance of seal formation, the tip of the pipette was first dipped into an antibiotic-free solution (140 mM KCl) for a few seconds and then back-filled with the antibiotic-containing solution. Once the whole-cell recording was established, the current-clamp mode (I = 0) was used for measurement of the membrane potentials. The membrane potentials of spermatogonia, spermatocytes, and early round spermatids were -45.66, -46.16, and -40.83 mV (n = 6), respectively.

Voltage- and Ca2+-Dependent Potassium Channels in Spermatogonia

Previous studies have characterized some of the voltage-dependent currents present in spermatogenic cells, most probably in the spermatocytes [7]; however, few studies have been conducted specifically on the spermatogonia and early spermatocytes. To detect voltage-dependent currents in the dissociated rat spermatogonia and early spermatocytes (from 15-day-old rat testes), an ensemble of voltage pulses ranging from -100 to +100 mV membrane potential was applied to cells clamped at a -70 mV holding potential in the whole-cell patch clamp configuration. We used 140 mM KCl as the pipette internal solution. As shown in Figure 1A, the predominant ionic current recorded from the spermatogonia and primary spermatocytes had an outwardly rectifying current-voltage relationship. These channels were observed in 45% of the cells from the 15-day-old rats studied (n = 75) but in none of the cells from the 80-day-old rats (n = 30). The potential at which channel opening first appeared was +40 mV. No active currents were detected if the membrane potential was less than +40 mV. The current increased dramatically when depolarization increased from +40 to +100 mV.



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  		FIG. 1. Whole-cell current traces and corresponding current-voltage (I-V) relationships in rat spermatogonia showing sensitivity to voltage and calcium ion. The internal solutions were 140 mM KCl (A) and 140 mM KCl plus 10 µM Ca2+ (B). The bath solution was Krebs Henseleit solution. The voltage protocol used consisted of a stepwise depolarization, in steps of 20 mV, from -100 mV to +100 mV with a holding potential of -70 mV. C) The relationship of current and voltage using a ramp test protocol by changing holding potential from -100 to +100 mV

To examine the Ca2+ dependence of this channel gating, we recorded macroscopic currents with protocols similar to that used in Figure 1A, but with 10 µM Ca2+ added to the pipette solution. Figure 1B shows that with 10 µM [Ca2+]i, the current amplitudes (420 ± 45 pA, n = 8) recorded were significantly greater than those (230 ± 37 pA, n = 8) recorded without Ca2+ added to the internal solution. The effect of increasing [Ca2+]i also shifted the gating voltage toward a more negative potential as shown in Figure 1C.

The activation of the current by [Ca2+]i was further confirmed by application of ionomycin, a calcium ionophore (Fig. 2, A–D). In this experiment, 1 mM ionomycin was added to the bath solution. This resulted in a doubling of the current amplitude from about 65 pA (Fig. 2A) to 130 pA (Fig. 2D) at a membrane potential of 100 mV, 1 min after ionomycin addition.



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  		FIG. 2. Effect of Ca2+ ionophore (ionomycin, 1 mM) on the current amplitude in a spermatogonium. The internal solution was 140 mM KCl. The voltage protocol was the same as that in Figure 1A. The bath solution was Krebs Henseleit solution containing 2.56 mM Ca2+. The current amplitude gradually increased on addition of ionomycin as shown from A (without ionomycin) to D (60 sec after ionomycin)

The K+ selectivity of the channel was examined by changing the K+ concentration in the bath. Figure 3 shows the current-voltage relationship of the currents measured at two different extracellular K+ concentrations. The concentration of KCl in the extracellular solution was raised from 6 to 70 mM by simultaneously reducing NaCl. Decreasing the outwardly directed K+ gradient attenuated the amplitude of the outwardly rectifying current, indicating that the current was K+ dependent.



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  		FIG. 3. Current-voltage relationship of the whole-cell current recorded under two different K+ gradients: 140 mM K+ intracellular and 6 mM K+ extracellular () and 140 mM intracellular and 70 mM K+ extracellular ({circ}). Continuous lines are the least-squares fits according to the Goldman-Hodgkin-Katz equation. In the case of a high extracellular K+ concentration, Na+ concentration in the bath solution was reduced accordingly to maintain ionic strength. Results are the mean ± SEM of three experiments

The effect of TEA, a known blocker of BKCa, on the spermatogonia whole-cell K+ current was investigated. The outwardly rectifying current was reduced when TEA (20 mM) was added to the internal pipette solution (Fig. 4, A and B).



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  		FIG. 4. Blockade of the outwardly rectifying currents by 20 mM TEA-Cl added to the pipette internal solution, which contained 140 mM KCl. Whole-cell current was studied at different voltages generated by stepwise depolarization, in steps of 20 mV, from -100 to +100 mV with a holding potential of -70 mV (A). The current-voltage (I-V) relationship was constructed using a ramp test protocol that changed holding potential from -100 to +100 mV within 180 msec.

Voltage-Dependent Potassium Currents in the Rat Spermatocytes

Figure 5 shows an outwardly rectifying current found in 80% of the cells from 80-day-old rats (n = 76). These cells were mainly secondary spermatocytes and spermatids. However, this current is different from the one recorded from spermatogonia in that its gating potential was about -40 mV (compared with +40 mV in spermatogonia). Furthermore, the current was inactivated when the membrane potential exceeded +60 mV (Fig. 5B). The activation of this channel exhibited time-dependent characteristics (Fig. 5A).



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  		FIG. 5. Characteristics of the outwardly rectifying currents present in spermatocytes and early spermatids. Whole-cell current recordings and corresponding current-voltage (I-V) relationships are shown in A and B, respectively. The internal solution contained 140 mM KCl, and the bath solution was Krebs Henseleit solution containing 2.56 mM Ca2+. The voltage protocol used consisted of a stepwise depolarization, in steps of 20 mV, from -100 to +100 mV with a holding potential of -70 mV. The ramp test protocol was used to change holding potential from -100 to +100 mV within 180 msec

Figure 6 illustrates the effect of 4-AP, a known Kv channel blocker, on the outwardly rectifying currents recorded in spermatocytes and early spermatids. When added to the bath solution, 5 mM 4-AP inhibited the currents by 52% ± 8% (n = 6) at a membrane potential of +60 mV.



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  		FIG. 6. Effect of 4-AP on Kv currents in spermatocytes. A) Current traces of a control cell. B) Current traces after external application of 5 mM 4-AP. C) Corresponding voltage-current relationships obtained from A and B. The activation protocol was the same as that in Figure 5

Switch of BKCa to Kv During Spermatogenesis

Thus far, we have recorded a BKCa channel mainly in spermatogonia and primary spermatocytes, and a Kv channel mainly in spermatocytes and spermatids. During spermatogenesis, germinal cells gradually change their morphology and membrane properties as they differentiate. A continuum of different channel expression should be found in some cells during developmental changes, although a particular channel population may exist only in certain phases and over a limited period of time. Figure 7 shows a spermatogonium exhibiting both the BKCa channels (activating voltages above +40 mV) and the Kv channels (activating voltages from -40 mV). The activating voltages are consistent with those reported previously in the germ cells obtained from 15- and 80-day-old rats, respectively (Figs. 1 and 5). The 30 pA whole-cell current activated between -20 and -40 mV was probably generated by the opening of the Kv channels. A single channel current of ~13 pA, probably due to the opening of a single BKCa channel, was seen superimposed on the whole-cell Kv current. Figure 8 shows the single channel properties of BKCa recorded under this condition. The single-channel conductance was 210 pS (picosiemens) at a membrane potential of +60 mV.



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  		FIG. 7. Two different potassium channels expressed in a single spermatogonium. The voltage protocol used consisted of a stepwise depolarization, in steps of 20 mV, from -100 to +100 mV with a holding potential of -70 mV. An outward whole-cell current (due to Kv channels) was observed from activating voltages of -40 mV. Intermittent current deflections superimposed on the whole-cell current when the cell was further depolarized to 40 mV were presumably caused by opening of BKCa channels (indicated by arrows)



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  		FIG. 8. Single channel properties of BKCa in a spermatogonium. Single channel openings induced by membrane depolarization to +60 mV in a spermatogonium studied under the conditions used in Figure 7. The line above the current trace in A represents the opened state of the channel. Opening to a conductance level of 13 pA was seen in this recording. B) Amplitude histogram constructed from the fitted data using Fetchan program. The 210-pS conductance is in agreement with the previous published value for BKCa

Detection of BKCa and Kv1.3 mRNAs in the Rat Spermatogenic Cells

The presence of BKCa and Kv1.3 mRNAs was detected with RT-PCR. Figure 9A shows the PCR products for the BKCa and Kv1.3 mRNAs in dissociated spermatogenic cells from immature (15-day-old) and mature (80-day-old) rats. The results show that BKCa mRNA was present in the germ cells of both 15- and 80-day-old rats; the Kv1.3 mRNA was detected in 80-day-old rat germ cells, but mildly so in 15-day-old rat germ cells. Sequences of the amplified BKCa and Kv1.3 fragments were blasted with the rat BKCa and Kv1.3 cDNA (at GenBank: http://www.ncbi.nlm.nih.gov/) and were found to match the sequence of rat BKCa and Kv1.3 cDNA (data not shown).



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  		FIG. 9. Detection of BKCa and Kv1.3 mRNAs and proteins in rat germ cells. See Results for explanation

Identification of BKCa and Kv1.3 Proteins in the Spermatogenic Cell Membrane

Figure 9, B and C, shows the results of Western blot analysis for BKCa and Kv1.3 proteins in the membrane fraction of germ cells isolated from 80-day-old rats. Using antibody against Kv1.3 (1:500 dilution; Alomone Labs), a 55-kDa immunoreactive band was detected in the membrane extract. However, a 122-kDa immunoreactive band was detected when antibody against BKCa (1/500 dilution; Alomone Labs) was used. These molecular weights are consistent with the calculated molecular weights for rat BKCa and Kv1.3, respectively.

Immunolocalization of Kv1.3 and BKCa Proteins in Mature Rat Testis

The localization of Kv1.3 and BKCa channels in the seminiferous tubules (stages VI–VIII) of mature rat testes was revealed by immunohistochemistry. The staining pattern was found to be germ cell-specific. Specific immunoreactivity for Kv1.3 channel was restricted to the plasma membrane of round spermatids (Fig. 10, B and C), whereas no immunoreactivity was detected in the spermatogonia and primary spermatocytes. In contrast, BKCa immunoreactivity was localized in the plasma membrane of primary spermatocytes and possibly also the spermatogonia (Fig. 10, E and F) and vascular smooth muscle cells known to contain such channel. No immunoreactivity of BKCa was detected in the spermatids (Fig. 10F).



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  		FIG. 10. Immunohistochemical localization of BKCa and Kv1.3 channel proteins in seminiferous tubules (stages VI—VIII) of a mature rat testis. The reddish peroxidase reaction product indicates the localization of the channels. Kv1.3 immunoreactivity was seen in the plasma membrane of the early spermatids (B and C). No immunoreactivity was detected in the spermatogonia. A) Control testis section in which the primary anti-Kv1.3 antibody was omitted. BKCa immunoreactivity was seen in the plasma membrane of spermatogonia and vascular smooth muscle cells (E and F). No immunoreactivity was detected in the spermatids. D) Control testis section in which the primary anti-BKCa antibody was omitted


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Approximately 100–200 million sperm are produced by a human testis daily. In generating this large number of spermatozoa, the spermatogonia must undergo cell division and differentiation to generate the haploid gametes, the spermatozoa. The control of cell proliferation and differentiation is likely influenced by external signals that modify membrane properties of the cells. Potassium channels have been shown to be involved in cell proliferation [25–33] and cell differentiation [34–38] in many cell types. The present finding that different K+ channels are expressed in different types of germ cells may indicate that these channels play distinct roles during the course of spermatogenesis.

Large conductance Ca2+-activated potassium channels (BKCa channels) comprise a large group of membrane proteins found in a wide variety of cells [39]. These channels are identified by their potassium selectivity and large single-channel conductance, as well as their ability to sense changes in both membrane voltage and intracellular Ca2+ concentration [40]. The predominant whole-cell current recorded in spermatogonia and primary spermatocytes is an outwardly rectifying K+ current. This current is apparently due to K+ channels having a single-channel conductance of 210 pS and is activated by membrane depolarization. This channel is also controlled by cytosolic Ca2+. Both direct application of Ca2+ (10 µM) into the cytosol or use of a Ca2+ ionophore (ionomycin) to increase intracellular Ca2+ resulted in an increase in the current amplitude. Reducing the pipette K+ concentration reduced the current amplitude, as expected for a K+-selective current. The presence of a large conductance Ca2+-activated K+ channel is supported by the presence of BKCa mRNA and proteins in the germ cells (Fig. 9). Immunohistochemistry also localized the protein in primary spermatocytes and possibly spermatogonia but not in postmeiotic germ cells (Fig. 10). Although the K+ channels described here are similar in conductance and gating properties to the "maxi-K" channel observed in many other tissues, the role of the 210-pS K+ channels in spermatogenesis is unknown. Day et al. [26] reported that a 240-pS K+ channel in the mouse early embryo is linked to the cell cycle: the channel is active throughout M and G1 phases, and switched off during the G1-to-S transition. The exact nature of the link between the spermatogonial cell proliferation and activity of the 210-pS K+ channel is not clear.

In addition to the BKCa which may control proliferation in nonexcitable cells, the small conductance voltage-gated potassium channels (Kv) have been identified in lymphocytes (T and B cells), Schwann cells, carcinoma cells of colon cancer, oligodendrocyte progenitor cells, microglia, and melanoma cells [25, 27–32], in which these channels may also control proliferation of the cells. K+ channel blockers inhibit proliferation of lymphocytes and of Schwann cells presumably by disrupting cell volume control [25, 31]. Unlike BKCa, a small conductance voltage-sensitive K+ channel sensitive to 4-AP is present in spermatocytes and spermatids but not in spermatogonia and primary spermatocytes. RT-PCR and Western blot analysis also supported the presence of Kv mRNA and Kv channel protein in the germ cells of mature rat testis (Fig. 9). Kv channel immunoreactivity was localized in the postmeiotic germ cells (Fig. 10). The role of the Kv channels in the spermatocytes and spermatids has yet to be clarified. The channels may regulate the resting membrane potential and/or intracellular Ca2+ and K+ concentrations, thereby controlling cell proliferation. They may also be involved in cell volume regulation during spermatogenesis.

Expression of BKCa is highest in the premeiotic germ cells but lowest in the postmeiotic germ cells, whereas the reverse is true for the Kv channel. It can be argued that since spermatogonia and primary spermatocytes were prepared from 15-day-old rat testes whereas spermatocytes and spermatids were prepared from 80-day-old rat testes, the difference in channel expression could have been due to the difference in age of the animals from which the germ cells were obtained, but not due to the difference in cell types. However, this possibility was precluded by the findings that the whole-cell current characteristics of spermatogonia obtained from immature rats were similar to those from mature rats (data not shown). It is tempting to speculate that a change from BKCa and Kv expression in spermatogenic cells is in some way linked to the course of spermatogenesis. The exact significance of the change is unknown, but since BKCa and Kv channels are normally subject to regulation by different agents, this phenomenon could allow the premeiotic and postmeiotic germ cells to be differentially regulated so that spermatogenesis can be carried out in a orderly and timely fashion.

Agents that block or open ion channels are useful in the study of the role of ion channels in spermatogenesis and fertility. In addition to the more conventional K+ channel blockers used in the present study, the more recently discovered animal toxins such as iberiotoxin (IBTX), which blocks BKCa [41], and the scorpion toxin charybdotoxin, which blocks Kv channels [42] with high specificity, could be useful tools for investigations. Although it is difficult to study the effects of ion channel blockers on the proliferation of germ cells in vitro, it is however possible to study their effects in vivo, taking caution that these drugs also block ion channels in other tissues. Such an approach has led to the discovery that the cystic fibrosis transmembrane conductance regulator, a cAMP-activated chloride channel, is involved in spermiogenesis as blockers of cystic fibrosis transmembrane conductance regulator (CFTR) inhibit spermiogenesis in the rat [43–45]. Lastly, useful information regarding ion channel function in reproductive function could also be obtained from transgenic animals in which specific ion channel genes are disrupted by gene targeting [13, 46]. Such information will be of paramount importance in our understanding of how ion channels work in spermatogenesis.


   FOOTNOTES
 
First decision: 10 December 2001.

1 This work was supported by a grant from the Rockefeller Foundation/Ernst Schering Research Foundation. Back

2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk Back

Accepted: January 29, 2002.

Received: November 19, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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