Calcium channels in chicken sperm regulate motility and the acrosome reaction

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1 Calcium channels in chicken sperm regulate motility and the acrosome reaction Thi Mong Diep Nguyen,,,, Anne Duittoz,,, Christophe Praud 5, Yves Combarnous,, and Elisabeth Blesbois,, Institut National de la Recherche Agronomique, UMR 85, Physiologie de la Reproduction et des Comportements, Nouzilly, France Centre National de la Recherche Scientifique, UMR 77, Nouzilly, France Universite Francßois Rabelais de Tours, Tours, France Faculty of Biology-Agricultural Engineering, Quy Nhon University, Quy Nhon, Vietnam 5 Institut National de la Recherche Agronomique, UR08 Recherches Avicoles, Nouzilly, France Keywords calcium channels; chicken sperm Correspondence E. Blesbois, Physiologie de la Reproduction et des comportements, INRA, CNRS, IFCE, Universite de Tours, 780 Nouzilly, France Fax: Tel: E mail: Elisabeth.Blesbois@tours.inra.fr (Received 0 December 05, revised February 06, accepted 9 March 06) doi:0./febs.70 Intracellular cytoplasmic calcium ([Ca ] i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca channel (SOC) component Orai, the transient receptor potential channel (TRPC) and inositol-,,5 trisphosphate receptors (IP R). L- and R type channels were mainly localized in the acrosome and the midpiece, and T type channels were not detected in chicken sperm. Orai was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP R was mainly detected in the nucleus. The L type channel inhibitor nifedipine, the R type channel inhibitor SNX 8 and the SOC inhibitors MRS 85, APB and YM 588 decreased [Ca ] i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T type channels may not be involved. In addition, Ca entry via SOCs appears to be the most likely pathway for AMPK activation and energyrequiring sperm functions such as motility and the acrosome reaction. Introduction In all eukaryotic cells, Ca signal transduction regulates a number of cellular functions, including transcription, enzyme activities, protein phosphorylation, cell death and many others []. In mammalian sperm, an increase in the concentration of intracellular cytosolic Ca ([Ca ] i ) is critical for several physiological Abbreviations [Ca ] I, intracellular cytoplasmic calcium; AMPK, AMP-activated protein kinase; AR, acrosome reaction; HVA, high voltage-activated; IP R, inositol,,5-trisphosphate receptor; LVA, low voltage-activated; Orai, calcium release-activated calcium modulator; SOCE, store-operated calcium entry; SOC, store-operated Ca channel; STIM, stromal interacting molecule; STR, straightness; TRPC, transient receptor potential channel; VAP, path velocity; VCL, curvilinear velocity; VOCC, voltage-dependent Ca channel; VSL, progressive velocity. 90 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

2 T.M.D. Nguyen et al. Ca channels in chicken sperm functions processes leading to fertilization, such as sperm motility and the acrosome reaction []. Increases in [Ca ] i may be generated either by release of Ca from intracellular stores or by influx of Ca across the plasma membrane. In many cases, these two processes are functionally coupled in order to replenish intracellular Ca stores and mediate long-term cytosolic Ca signals []. In most cells, entry of external Ca occurs through several types of Ca channels: mainly voltage-activated channels [] and store-operated channels [5]. Ca may be released from internal stores via receptor-operated channels. Inositol-,,5-trisphosphate (IP ) receptors (IP R) and ryanodine receptors represent the two main intracellular Ca channels responsible for releasing stored Ca. In the sperm of many animal classes, members of a specific transmembrane Ca channel family, CatSper, also play an important role [6]. The voltage-dependent Ca channels (VOCCs) belong to a family of transmembrane proteins [7], and play an important role in ion influx into sperm []. The major function of VOCCs is to convert changes in membrane potential into intracellular signal involving Ca. The VOCCs are classified into two functional classes: high voltage-activated channels (HVA) and low voltage-activated channels (LVA). These are classified according to the a subunit: the HVA channels include L type channels Ca v. (a S ), Ca v. (a C ), Ca v. (a D ) and Ca v. (a F ), the P/Q type channel Ca v. (a A ), the N type channel Ca v. (a B ), and the R type channel Ca v. (a E ); the LVA channels include the T type channels Ca v. (a G ), Ca v. (a H ) and Ca v. (a I ) [8]. VOCC activities have been detected in mammalian and sea urchin germ cells and sperm [9,0]. The localization of various VOCCs in the head and the flagellum of sperm suggests that they are involved in modulation of motility and the acrosome reaction (AR), two important events that require Ca. Unlike VOCCs, store-operated Ca channels (SOCs) are voltage-independent Ca channels that are activated in somatic cells upon depletion of Ca stores in the endoplasmic reticulum (ER), and triggered physiologically through stimulation of a diverse set of surface receptors. Since sperm do not contain ER, other internal stores have been reported to be involved, such as the acrosome vesicle [,]. Two protein families, Orai (calcium release-activated calcium modulators) and STIM (stromal interacting molecule), have emerged as candidates for components of store-operated calcium entry (SOCE) []. In the Orai family, Orai (Orai calcium release-activated calcium modulator ) was identified as a main protein of the SOC complex []. In somatic cells, the activity of Orai is closely controlled by the ER membrane protein stromal interacting molecule (STIM), which functions as an ER Ca sensor [5]. SOCs have also been suggested to be encoded by the transient receptor potential channel TRPC [6,7], and the conformational coupling of TRPC to the IP R [8,9] or other components of the ER may be responsible for SOCE activation [7]. TRPC may also combine or interact with Orai and STIM to form and regulate store-operated channels [0,]. Mammalian sperm possess SOCs, which have been proposed to be responsible for the sustained [Ca ] i elevation necessary for the AR [,,]. In human sperm, Orai is primarily localized in the acrosome and midpiece and to a lesser extent on the principal piece, and STIM is localized in the midpiece and the neck [,5]. TRPC was found exclusively in the flagellum in the sperm of mice [6] and humans [7]. The intracellular receptor IP R is a calcium channel that permits efflux of Ca sequestered in intracellular stores into the cytoplasm. Three isoforms of IP R have been identified in mammals: IP R IP R. In germinal cells, selective localization and functional association of IP Rs with the acrosome has been reported [8,9]. The acrosome, which includes a vesicle that contains different hydrolytic enzymes, also plays a role as a Ca store [,]. The IP R was also found at the head and the flagellum in humans, bovine, rat, hamster, mouse and dog sperm [,,8 ]. Use of an IP R activator, thimerosal, triggered the AR in mouse sperm, which suggests that the release of Ca from intracellular reservoirs via IP R is an important step in the AR process in mammalian sperm []. The CatSper channels have been suggested to be very important for regulation of motility and the AR in mammalian sperm [6]. However, the CatSper genes are not functional due to the presence of many stop codons in vertebrate lineages such as Agnatha, Teleostei, Amphibia and Aves [], and are thus one of the gene functionalities lost during avian evolution. The presence and activity of Ca channels involved in mammalian sperm functions have already been the subject of numerous studies. To our knowledge, despite the importance of Ca in the regulation of motility and the AR in birds [ 6], the whole range of sperm Ca channels have never been taken into consideration in avian species. The present study investigated the presence, distribution and effects of VOCCand SOC-related proteins in chicken sperm and their relative involvement in sperm motility and the AR. These studies of VOCCs, Orai, TRPC and IP R, which are the main candidates for involvement in liberation of intracellular calcium stores, are an initial step to assess the structural elements of Ca entry The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 90

3 Ca channels in chicken sperm functions T.M.D. Nguyen et al. channels in these cells, which are involved in the complex oviparous internal fertilization system specific to birds. These channels were chosen because of their importance in sperm of other animal classes (HVA L- and R type VOCCs, LVA T type VOCCs and IP R), or because of their putative role (Orai and TRCP) as components of SOCs. Finally, we also assessed potential effects of VOCCs and SOCs on a key player in regulation of energetic metabolism, AMP-activated protein kinase (AMPK), which is known to be central to sperm functions [7]. Results Identification and localization of calcium channels in chicken sperm The presence of the L type (Ca v.), R type (Ca v.) and T type (Ca v./. and Ca v.) channels and Orai, TRPC and IP R in chicken sperm was assessed by Western blotting and indirect immunofluorescence staining using primary antibodies against Ca v., Ca v., Ca v./., Ca v., Orai, TRPC and IP R. VOCCs (L type, R type and T type channels) Western blotting of the L-type channel (Ca v.) showed the expected 05 kda band in chicken sperm as well as in rat skeletal muscle (control) (Fig. A). Immunostaining showed that the L type channel is localized mainly in the acrosome and the midpiece of chicken sperm (Fig. E). Western blotting for the R type channel (Ca v.) revealed a main band with an apparent molecular mass of 57 kda in chicken sperm as well as in the rat testis (control) (Fig. A). There was strong immunostaining for the R type channel in the acrosome and the midpiece, but much lower fluorescence in the flagellum (Fig. E). By contrast, the T type channels (Ca v./. and Ca v.) were not detected in chicken sperm, either by immunofluorescence or by Western blotting, although the expected 6 kda protein was present in the rat testis (control) (Fig. A,B). SOC candidate proteins (Orai and TRPC) Western blotting for Orai in chicken sperm as well as in rat testis revealed a main band at a molecular mass of 50 kda (Fig. A), although the predicted band size is kda. The actual band size was most probably shifted to a higher size due to homodimerization of Orai monomers [8] or possible post-translational modifications (O glycosylation of Ser or Thr residues cannot be ruled out) as reported by the antibody manufacturer (Abcam, Paris, France). Orai was also strongly detected in the head of the sperm (which contains the acrosome and the nucleus) and the midpiece, with a much lower signal in the flagellum (Fig. E). A protein of the predicted size (9 kda) corresponding to TRPC (Fig. 5A) was detected by Western blotting in chicken sperm as well as in rat skeletal muscle (control). Strong immunoreactivity for TRPC was observed in the acrosome and midpiece regions (probably in the plasma membrane), with a much lower signal in the nucleus area and the flagellum (Fig. 5E). IP R A band at kda, corresponding to the predicted mass of IP R, was found by Western blotting in chicken sperm as well as in rat skeletal muscle (control) (Fig. 6A). Immunofluorescence data showed that the IP R was present mainly in the nucleus area of chicken sperm but not in the acrosome (Fig. 6E). Effects of calcium channel inhibitors on intracellular Ca in chicken sperm The intracellular free Ca concentration in intact sperm, calculated before addition of 5 mm extracellular Ca, was approximately 5 nm at 5 C (Fig. 7). After addition of extracellular Ca, the intracellular Ca concentration increased rapidly and reached a plateau at approximately 50 nm (Fig. 7). The influx of Ca through the plasma membrane was significantly affected by nifedipine (an L type channel inhibitor), SNX 8 (an R type channel inhibitor) and MRS 85 (an SOC inhibitor) compared to the positive control (with Ca and no inhibitor) (Fig. 7A). However, the effect of MRS 85 on the influx of Ca was significantly stronger than that of nifedipine (P < 0.005) but significantly smaller than that of SNX 8 (P < 0.0) or the three inhibitors combined (P < 0.0). The effect of nifedipine on the influx of Ca was significantly smaller than that of SNX 8 (P < 0.00). There was a significant difference in Ca influx between treatment with the mixture of the three inhibitors and the negative control (without Ca and no inhibitor) in the period up to 000 s (P < 0.0). The mixture of the three inhibitors almost completely abolished Ca entry from the medium after 000 s, and therefore there was no significant difference relative to the negative control after this time. We performed another experiment with two other SOC inhibitors ( APB and YM 588), which gave 90 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

4 T.M.D. Nguyen et al. Ca channels in chicken sperm functions A kda Ca v. B Transmission Fig.. Identification and localization of the L type channel in chicken sperm. (A) Protein from chicken sperm lysates was analyzed by Western blotting using antibody against Ca v.. Cell lysates from rat skeletal muscle were used as a positive control. A band of approximately 9 kda was detected. (B) Indirect immunofluorescence staining of chicken sperm was performed using the same antibody. White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). (C) Nuclei were stained blue using DAPI. (D) Immunofluorescence staining of the L type channel (Ca v.) (green). (E) Merged images of fluorescence staining. White arrows in (D) and (E) indicate regions showing L type channel (Ca v.) immunoreactivity Chicken sperm Rat skeletal muscle DAPI Ca v. C D Merge (Fluorescence) E A kda B Transmission Fig.. Identification and localization of the R type channel in chicken sperm. (A) Protein from chicken sperm lysates was analyzed by Western blotting using antibody against Ca v.. Cell lysates from rat testis were used as a positive control. A band of approximately 57 kda was detected. (B) Indirect immunofluorescence staining of chicken sperm was performed using the same antibody. White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). (C) Nuclei were stained blue using DAPI. (D) Immunofluorescence staining of the R type channel (Ca v.) (green). (E) Merged images of fluorescence staining. White arrows in (D) and (E) indicate showing R type channel (Ca v.) immunoreactivity. 50 Ca v Rat testis Chicken sperm DAPI Ca v. C D E Merge (Fluorescence) the same results as those obtained using MRS 85. The [Ca ] i was significantly decreased in response to APB and YM 588 compared to the positive control (with Ca and no APB and YM 588) (P < 0.005) (Fig. 7B). There was no significant difference in [Ca ] i between APB and YM 588 treatments (P = 0.09). Effects of L type, R type and SOC inhibitors on sperm motility, viability and the AR Sperm motility A 5 min incubation in the presence of Ca channel inhibitors led to significant reductions in motile sperm The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 905

5 Ca channels in chicken sperm functions T.M.D. Nguyen et al. A kda Rat testis a Chicken sperm 50 Ca v./ Transmission 50 DAPI Ca v./. Merge (Fluorescence) b c d e B kda a Rat testis Chicken sperm Ca v. Transmission Ca v. b d DAPI Merge (Fluorescence) c e Fig.. Identification and localization of the T type channel in chicken sperm. Western blotting of sperm proteins using the anti- Ca v./. and anti-ca v. antibodies. Cell lysates from rat testis were used as a positive control. (A) A band of approximately 6 kda was detected using the antibody against Ca v./. (a). Indirect immunofluorescence staining of chicken sperm was performed using the same antibody (b). White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). Nuclei were stained blue using DAPI (c). No green signal corresponding to Ca v./. was observed (d). Merged images of fluorescence staining are shown in (e). (B) A band of approximately 7 kda was detected using the antibody against Ca v. (a). Indirect immunofluorescence staining of chicken sperm was performed using the same antibody (b). White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). Nuclei were stained blue using DAPI (c). No green signal corresponding to Ca v. was observed (d). Merged images of fluorescence staining are shown in (e). compared to the positive control (with Ca and no inhibitor) (P < 0.05) whatever the inhibitor used, and led to suppression of the stimulation of Ca influx (Table ). There was no significant difference in the effects on sperm motility between the L type channel inhibitor (nifedipine), the R type channel inhibitor (SNX 8) and the SOC inhibitor (MRS 85). After 0 min, motile sperm were still significantly decreased in the presence of Ca channels inhibitors compared to the positive control (Table ). MRS 85 suppressed the effect of extracellular Ca more efficiently than nifedipine, while SNX 8 gave intermediate results. MRS 85 was almost as efficient as the three inhibitors together (nifedipine + SNX 8 + MRS 85). The inhibition of all three channel types was the most efficient in decreasing motile sperm (50% decrease). The same progression was observed for progressive sperm and rapid sperm at 5 and 906 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

6 T.M.D. Nguyen et al. Ca channels in chicken sperm functions A kda B Transmission Fig.. Identification and localization of Orai in chicken sperm. (A) Protein from chicken sperm lysates was analyzed by Western blotting using antibody against Orai. Cell lysates from rat testis were used as a positive control. A band of approximately 50 kda was detected. (B) Indirect immunofluorescence staining of chicken sperm was performed using the same antibody. White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). (C) Nuclei were stained blue using DAPI. (D) Immunofluorescence staining of Orai (green). (E) Merged images of fluorescence staining. White arrows indicate regions showing Orai immunoreactivity D Rat testis Chicken sperm Orai Orai C E Merge (Fluorescence) DAPI Fig. 5. Identification and localization of TRPC in chicken sperm. (A) Protein from chicken sperm lysates was analyzed by Western blotting using antibody against TRPC. Cell lysates from rat testis were used as a positive control. A band of approximately 9 kda was detected. (B) Indirect immunofluorescence staining of chicken sperm was performed using the same antibody. White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). (C) Nuclei were stained blue using DAPI. (D) Immunofluorescence staining of TRPC (green). (E) Merged images of fluorescence staining. White arrows indicate regions showing TRPC immunoreactivity. A kda DAPI Chicken sperm Rat skeletal muscle TRPC C D E TRPC B Transmission Merge (Fluorescence) 0 min (Tables and ). After 0 min, the motility levels obtained in response to the mixture of the three inhibitors were lower than those obtained in the absence of extracellular calcium, suggesting disturbance in the release of intracellular calcium stores. The other sperm motility parameters analyzed were also reduced in the presence of inhibitors. The curvilinear velocity (VCL), progressive velocity (VSL) and path velocity (VAP) were all significantly reduced in the presence of the inhibitors in a time-dependent The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 907

7 Ca channels in chicken sperm functions T.M.D. Nguyen et al. A kda DAPI Rat skeletal muscle Chicken sperm IP R C D E IP R B Transmission Merge (Fluorescence) Fig. 6. Identification and localization of IP R in chicken sperm. (A) Protein from chicken sperm lysates was analyzed by Western blotting using antibody against IP R. Cell lysates from rat testis were used as a positive control. A band of approximately kda was detected. (B) Indirect immunofluorescence staining of chicken sperm was performed using the same antibody. White arrows indicate areas of the acrosome (arrow ), the nuclei (arrow ), the midpiece (arrow ) and the flagellum (arrow ). (C) Nuclei were stained blue using DAPI. (D) Immunofluorescence staining of IP R (green). (E) Merged images of fluorescence staining. White arrows indicate regions showing IP R immunoreactivity. manner, but with some differences between the parameters measured. At 5 min, nifedipine reduced VCL but not VAP or VSL. SNX 8 significantly affected VCL and VSL but not VAP, while MRS 85 decreased all three parameters (Table ). After 0 min, most VAP, VCL and VSL values were decreased compared to the positive control, and the effects of individual inhibitors were abolished. The effect of treatment with the combination of inhibitors on VCL was maintained (Table ). Given the results obtained for use of the SOC inhibitor MRS 85, we also studied the effect of two additional SOC inhibitors ( APB and YM 588). APB and YM 588 affected cell motility in a timedependent fashion, decreasing motile sperm by 7% for APB and % for YM 588 at 5 min, and by % for both at 0 min, compared to the positive control (with Ca and no inhibitor) (P < 0.05) (Fig. 8). As for MRS 85, APB and YM 588 abolished the stimulation of Ca influx at 5 and 0 min. Moreover, after 0 min incubation, motile sperm in the negative control without extracellular Ca were lower than at 5 min, suggesting added intracellular disturbances (Fig. 8). causes a significant decrease in AR, stronger that that resulting from treatment with nifedipine or SNX 8, after 5 and 0 min incubation (P < 0.05). Nifedipine significantly decreased the AR compared to SNX 8 at 5 min (P < 0.05) but not at 0 min. Simultaneous use of the three inhibitors almost completely eliminated the AR, suggesting almost total blocking of all calcium channels, including intracellular calcium channels. Moreover, the ability of sperm to undergo the AR decreased over time in all treatments. We observed a significant decrease in the AR after 0 min incubation for both controls, with or without Ca, as well as for the treated samples. To further investigate the involvement of SOCs in the AR in chicken sperm, cells were incubated in the presence of APB and YM 588, and AR was determined at various times. The results revealed that APB and YM 588 caused a significant decrease in AR at 5 and 0 min compared to either control. There was no significant difference in the AR between APB and YM 588 (Fig. 9B). The values obtained in the presence of APB and YM 588 were similar to those observed in cells incubated for the same period of time with MRS 85. Sperm acrosome reaction The sperm AR was extremely reactive to blocking of Ca channels by inhibitors, rapidly resulting in AR levels that were significantly lower than in either control (Fig. 9A). The results revealed that MRS 85 Sperm viability The sperm viability was equivalent between the controls and all inhibitory treatments during the first 0 min. A significant decrease was observed after 0 min incubation for the controls as well as for the samples treated 908 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

8 T.M.D. Nguyen et al. Ca channels in chicken sperm functions A [Ca ] i (nm) mm Ca *** *+ * *+ ns Times (s) + + * * * Ctrl (5 mm Ca added) Ctrl (No added Ca ) Ca + 0 µm Nifedipine Ca + 60 nm SNX-8 Ca + 0 µm MRS-85 Ca + Nifedipine + SNX-8 + MRS-85 B 5 mm Ca 00 Ctrl (5 mm Ca added) 00 Ctrl (No added Ca ) [Ca ] i (nm) ns * * * Ca +50 µm -APB Ca +00 nm YM Times (s) Fig. 7. Changes in the intracellular free Ca concentration in chicken sperm with or without inhibitors of Ca channels at 5 C. Sperm cells were incubated with Fluo AM as described in Experimental procedures. The intracellular Ca levels ([Ca ] i ) were analyzed by spectrofluorimetry using an excitation wavelength of 9 nm and an emission wavelength of 56 nm. [Ca ] i measurements were made every 50 s. Baseline levels of [Ca ] i were monitored for 500 s prior to adding 5 mm Ca. After injection of Ca changes in [Ca ] i were monitored for an additional 500 s. (A) Effect of Ca channel activity inhibitors on [Ca ] i. Sperm were incubated in the presence of 5 mm Ca and were pre-treated with 0 lm nifedipine, 60 nm SNX 8, 0 lm MRS 85, or nifedipine + SNX 8 + MRS 85. The positive control comprised addition of 5 mm Ca and no Ca channel inhibitor; the negative control comprised no added Ca and no Ca channel inhibitor. Values are means SEM. Each point on the curve represents the mean of six repetitions with three sperm samples per repetition for each sample. Asterisks and plus symbols indicate statistically significant differences between positive control and nifedipine, MRS 85, SNX 8, or the mixture of the three inhibitors (***P < 0.005; +P < A significant difference was found between MRS 85 and nifedipine (***P < 0.005) or SNX 8 (*+P < 0.0) or the mixture of the three inhibitors (*+P < 0.0). A significant difference between nifedipine and SNX 8 (+P < 0.00) was also found, as well as between the three inhibitors combined and the negative control (*+P < 0.0, from 500 to 000 s). There was no significant difference between the three inhibitors and the negative control after 000 s. (B) Sperm incubated in the presence of 5 mm Ca and 50 lm APB or 50 lm YM 588. The positive control comprised addition of 5 mm Ca and no Ca channel inhibitor; the negative control comprised no added Ca and no Ca channel inhibitor. Values are means SEM. Each point on the curve represents the mean of four repetitions with three sperm samples per repetition for each sample. A significant difference was found when comparing the positive control against APB or YM 588 treatments or the negative control (*P < 0.005). There was no significant difference in [Ca ] i between APB and YM 588 treatments. with Ca channel inhibitors (Fig. 0). There was no effect of the inhibitors on sperm viability. Effects of L type, R type and SOC inhibitors on AMPK phosphorylation We measured AMPK phosphorylation in the presence of Ca channel inhibitors. Addition of the SOC inhibitor MRS 85 suppressed the increase in AMPK phosphorylation induced by extracellular Ca compared to the positive control (with Ca and no inhibitor) (P < 0.05) (Fig. ). The L type channel inhibitor (nifedipine) and the R type channel inhibitor (SNX 8) tended to reduce the increase of AMPK phosphorylation induced by extracellular Ca compared to the positive control, but not significantly. Use The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 909

9 Ca channels in chicken sperm functions T.M.D. Nguyen et al. Table. Effect of Ca channel inhibitors on motility parameters of chicken sperm after 5 min incubation. Sperm were incubated for 5 min at 0 C in Lake 7. medium with or without 5 mm Ca in the presence or absence of Ca channel activity inhibitors (nifedipine, SNX 8, MRS 85, or nifedipine + SNX 8 + MRS 85). Values represent the mean SEM of six experiments. Superscripts indicate significant within-row differences (P < 0.05). 5mM Ca added, plus Motility parameters Negative control (no added Ca ) Positive control (5 mm Ca added) 0 lm nifedipine 60 nm SNX 8 0 lm MRS 85 Nifedipine + SNX 8 + MRS 85 Motile (%) 5..9 b 6.0. a b.0.9 bc bc c Progressive (%) 8..0 b 9..7 a 8.. b.8.0 bc.8.0 bc.5.7 c Rapid (%).. bc 0.. a 7..7 b b.0.6 bc 9.. c VAP (lms ) b a ab ab b b VCL (lms ) 8.5. b a..0 b.8.7 b.8.9 b.0.7 b VSL (lms ).0. b a 7.5. ab.9. b 5.0. b.6. b Table. Effect of Ca channel inhibitors on motility parameters of chicken sperm after 0 min incubation. Sperm were incubated for 5 min at 0 C in Lake 7. medium with or without 5 mm Ca in the presence or absence of Ca channel activity inhibitors (nifedipine, SNX 8, MRS 85, or nifedipine + SNX 8 + MRS 85). Values represent the mean SEM of six experiments. Superscripts indicate significant within-row differences (P < 0.05). 5mM Ca added, plus Motility parameters Negative control (no added Ca ) Positive control (5 mm Ca added) 0 lm nifedipine 60 nm SNX 8 0 lm MRS 85 Nifedipine + SNX 8 + MRS 85 Motile (%).8.0 c a 0..7 b.7. bc.. cd.9.5 d Progressive (%).. bc.0.5 a 8.7. ab..9 bc.0. bc c Rapid (%) c 5..8 a b bc c.8.5 c VAP (lms ) a a a a 58.. a a VCL (lms ) 5.8. b.7.7 a..9 ab 8..9 ab 7..8 ab b VSL (lms ).8. a a.0.6 a 5..7 a.. a.. a of the three inhibitors together gave results equivalent to those for MRS 85 alone, showing the importance of SOCs in the rapid Ca -dependent increase in AMPK phosphorylation. Discussion Various studies in avian sperm have indicated that an increase in extracellular Ca is critical for several physiological processes leading to successful fertilization, such as sperm motility and the AR [ 6]. However, little was known about which pathways regulate these events and how it was done. Most studies on sperm Ca -permeable channels have been performed in mammals or in sea urchins, and growing evidence indicates significant differences between the animal classes [6,9,0, ]. The findings presented here provide what is to our knowledge the first evidence for the presence or absence of various VOCCs (L type, R type and T type) and SOCs (Orai and TRPC), as well as IP R, in avian sperm, and their possible involvement in the regulation of sperm motility and AR capacity. We present evidence for the presence and expression of HVA L type and R type VOCCs but not LVA T type VOCCs (Ca v./ca v. and Ca v.) in chicken sperm. In mammalian and sea urchin sperm, multiple VOCCs were detected, including both HVA and LVA types [9]. Differences in the mechanisms of activation and inactivation of these channels provide a means for selective activation [0]. The channels are organized by type and activity, and are clustered into functional groups, possibly interacting through local effects on membrane potential []. The L type channel Ca v. was found in sea urchin flagella [], in the acrosome and the flagellum main piece in mice [], in the post-acrosome region and in the main piece of the flagellum in humans [], in the post-acrosome region and the flagellum in rat [5], and in the neck and principal piece of the tail in horse sperm [6]. The R type channel Ca v. was found in the mitochondria and the acrosome in sea urchin [], 90 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

10 T.M.D. Nguyen et al. Ca channels in chicken sperm functions % b Ctrl (No added Ca ) c Ctrl (5 mm Ca Motility a ab b b added) Ca + 50 µm -APB Ca 5 min 0 min d + 00 nm YM-588 Fig. 8. Effect of Ca channel inhibitors ( APB and YM 588) on sperm motility. Sperm were incubated in Lake 7. medium at 5 C in the presence of 5 mm Ca and 50 lm APB or 50 nm YM 588 for 5 min (green) or 0 min (red). The positive control comprised sperm incubated with 5 mm Ca and no Ca channel inhibitor. The negative control comprised sperm incubated in the absence of 5 mm Ca and no Ca channel inhibitor. Values are means SEM (n = 6). Different letters above bars indicate values that were statistically significantly different at P < d in the flagellum in humans [], and in the acrosome and the main piece in mice []. The T type channel Ca v. was found in the acrosome and the main piece in mice, and in the head region and the flagellum in humans [], while Ca v. was found in the mouse acrosome and main piece, and the main piece in humans [], and Ca v. was found in the mice midpiece and in the head region and the flagellum in humans []. At the functional level, antagonists of L type channels prevent the calcium increase and inhibit the AR in horse sperm [6]. The Ca v and Ca v channels have also been suggested to participate in human sperm capacitation and the AR [7]. In the AR, these channels are activated after binding of the sperm to the zona pellucida, and cause depolarization of the membrane [8]. In our study, the L type Ca v. and R type Ca v. channels were found in the acrosome and the midpiece, and also in the flagellum for the R type channel. We also showed that the L type channel inhibitor nifedipine and the R type channel inhibitor SNX 8 both affected intracellular Ca as well as sperm motility and the AR. Thus, based on the proposition that Ca coordinates mitochondrial ATP production with cellular energy demand and is a key regulator of sperm motility and AR [9 5], the HVA L type (Ca v.) and R type (Ca v.) VOCCs may be involved in an increase in calcium influx in the acrosome and midpiece of chicken sperm, and play a primary role in the AR and sperm motility. The presence of R type but not L type channels in the flagellum suggests that the R type channel has a more direct role than the L type channel on flagellar movement. Although it has been deduced from the chicken genome that T type channels are present in these animals [5] (Table ), and they have been found to be present in chicken neurons [5], we did not observe them in chicken sperm by using Western blotting or immunocytochemistry. This is not surprising for Ca v. and Ca v. as the antibody homology was low (50%, Table ), but it is for Ca v. as its antibody showed good homology (8%). Furthermore, the voltagegated calcium channel isoleucine glutamine motif was not conserved in chicken T type channel sequences (Table ). We thus suggest that there is a definite absence of expression of the T type channel Ca v. in chicken sperm, while no clear answer may be given for Ca v. and Ca v.. This difference in expression between chickens and mammals may be related to differences in the sperm maturation process between animal classes, as chicken sperm do not undergo capacitation before the AR, a process that involves the three T type channels in mammals [5,6,0,,8]. Chicken sperm may remain for a very long time (up to weeks) in the female genital tract before reaching the site of fertilization and undergoing the AR to penetrate the perivitelline layer surrounding the oocyte. In contrast to mammals, the AR in birds may be induced very rapidly in vitro (in < 5 min) using a simple saline medium containing Ca and perivitelline layer [5 7]. In addition, calcium influx through HVA channels (R type and/or L type) is required for this rapid process. We thus hypothesize that LVA activation (T type Ca v channels) is not necessary for the chicken sperm AR, unlike in humans [7,8], and is replaced by R type channel activity, as it has been shown that the permeation properties of Ca v. closely resemble those described for a subset of low-threshold Ca channels [5]. However, this hypothesis needs to be refined due to the limitations of the antibodies and because it is unlikely that a single mechanism exists and other L type channel isoforms or other channels such as N type or P/Q type channels remain to be studied. In the present work, we provide the first evidence for the presence of SOCs in chicken sperm. A common observation in somatic cells is that mobilization of stored Ca leads to a decrease in [Ca ] i inside the storage organelle that causes activation of Ca influx through SOCs at the plasma membrane. The SOC candidates involved in somatic cells were previously The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 9

11 Ca channels in chicken sperm functions T.M.D. Nguyen et al. A % Viability a a a a a a b b b b b b 5 min 0 min % B Viability a a a a b b b b 5 min 0 min 0 0 Ctrl (No added Ca ) Ctrl (5 mm Ca added) Ca + 0 µm Nifedipine Ca + 60 nm SNX-8 Ca 0 Ca + 0 µm MRS-85 + Nifedipine+SNX-8MRS-85 0 Ctrl (No added Ca ) Ctrl (5 mm Ca added) Ca + 50 µm -APB Ca + 00 nm YM-588 Fig. 9. Effect of Ca channel inhibitors on sperm AR. Sperm were incubated in Lake 7. medium at 5 C in the presence of 5 mm Ca and (A) 0 lm nifedipine, 60 nm SNX 8 or 0 lm MRS 85, or (B) 50 lm APB or 50 nm YM 588 for 5 min (green) or 0 min (red). The positive control comprised sperm incubated with 5 mm Ca and no Ca channel inhibitor. The negative control comprised sperm incubated in the absence of 5 mm Ca and no Ca channel inhibitor. Values are means SEM (n = 6). Different letters above bars indicate values that were statistically significantly different at P < thought to be transient receptor potential channels, in particular TRPCs [55]. In the TRPC family, TRPC was the best candidate for involvement in SOC complexes [6,7] as endogenous TRPC has been shown to contribute to SOCE in several cell types [7]. However, since 005, numerous studies have demonstrated the involvement of two other transmembrane protein groups, Orai and STIM, that reside in the plasma membrane and the ER membrane, respectively, and demonstrate cross-talk that enables them to orchestrate successful SOCE [ 5]. The occurrence of SOCE has been demonstrated in mammalian and sea urchin sperm following Ca store mobilization [,5 7,55 57]. We also identified the transmembrane protein Orai for the first time in avian sperm. This is a major SOC component that forms the Ca pores. Of the Orai family, Orai is predicted to be the most important []. Orai in the plasma membrane is activated by STIM, an intracellular store calcium sensor, to mediate SOCE [5]. In this study, we were unable to confirm the presence of an STIM channel in chicken sperm due to the lack of suitable antibodies in birds. However, because the Orai/STIM interaction appears to be well conserved through the evolution, we suspect that STIM is also present in birds. We demonstrate here for the first time that Orai is expressed mainly in the acrosome, nucleus area and midpiece, and at a lower level in the flagellum of chicken sperm. Moreover, in this study, positive immunolocalization of TRPC was also shown in all compartments of chicken sperm instead of being confined to the flagellum as in mice and humans [6,7], suggesting its possible involvement in different sperm functions. Interestingly, and consistent with the regions of Ca storage, immunocytochemistry for Orai and TRPC revealed bright staining at the midpiece, the nucleus area (mainly for Orai) and the acrosome, suggesting that Orai and TRPC proteins are ideally positioned within chicken sperm to interact and promote SOCE as observed in other cell types [0,]. We also showed that Ca influx from the extracellular space was more strongly blocked by the SOC inhibitor MRS 85 than by L- or R type channel inhibitors, leading to inhibition of sperm motility and the AR. The importance of SOCs was confirmed by the use of two other SOC inhibitors: APB and YM 588. YM 588 directly interacts with SOCs or channel- 9 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

12 T.M.D. Nguyen et al. Ca channels in chicken sperm functions % A Acrosome reaction a b b c c de d e e fg f g 5 min 0 min B % Acrosome reaction a b b c d e cd e 5 min 0 min 0 Ctrl (No added Ca ) Ctrl (5 mm Ca added) Ca + 0 µm Nifedipine Ca + 60 nm SNX-8 Ca + 0 µm MRS-85 Ca + Nifedipine+SNX-8MRS-85 0 Ctrl (No added Ca ) Ctrl (5 mm Ca Ca added) + 50 µm -APB Ca + 00 nm YM-588 Fig. 0. Effect of Ca channel inhibitors on sperm viability. Sperm were incubated in Lake 7. medium at 5 C in the presence of 5 mm Ca and (A) 0 lm nifedipine, 60 nm SNX 8 or 0 lm MRS 85, and (B) 50 lm APB or 50 nm YM 588 for 5 min (green) or 0 min (red). The positive control comprised sperm incubated with 5 mm Ca and no Ca channel inhibitor. The negative control comprised sperm incubated in the absence of 5 mm Ca and no Ca channel inhibitor. Values are means SEM (n = ). Different letters above bars indicate values that were statistically significantly different at P < regulatory co-factors at the membrane surface without depleting Ca stores [57], and APB modulates the interaction of STIM with SOC subunits (Orai and possibly TRPC) in the plasma membrane [58,59]. The decreases in Ca influx, motility and the AR in the presence of APB and YM 588 were similar to those found with MRS 85. This indicates that the stimulating action of extracellular Ca on chicken sperm motility is mainly mediated by calcium entry through SOCs, rather than through VOCCs (L type and R type channels). The intracellular calcium increase necessary to trigger the AR has to be rapid, and decreasing the rate of calcium entry has been reported to abolish the AR [56]. Our results give support to this assumption, as extracellular calcium plays a central role in the chicken AR, which is induced in < 5 min in vitro [6]. We also hypothesize that, in chicken sperm, activation of SOCs is required for both the AR and motility. In addition, simultaneous inhibition of the three channel types (L type, R type and SOCs) produced the largest decrease in motility and the AR. The IP R is known to play a role that is coordinated with the SOC action. It is activated by binding of IP. This is the product of degradation of phosphatidylinositol bisphosphate by phospholipase C in response to stimulation of the G protein-coupled receptor or the tyrosine kinase receptor [60]. By binding to the IP R, IP allows release of calcium contained in intracellular stores. The level of IP binding in sperm is among the highest observed in mammalian tissues. In chicken sperm, IP R is mainly localized in the nucleus area of chicken sperm rather than the acrosome, midpiece or flagellum. These results differ from those obtained with mammalian sperm, as IP R was found in the acrosome and neck of human sperm [0,]. The localization of IP R in the chicken sperm nucleus area suggests that IP R contributes significantly to calcium influx in this part of the cell, which leads us to suggest that the nucleus area is an important Ca store. Based on its central localization between the acrosome and midpiece, it is likely that this store may be used for Ca signaling in other sperm compartments such as the acrosome and midpiece, and IP R may contribute significantly to calcium signaling during the AR and other metabolic functions of sperm. In addition, the observation of Orai, TRPC and IP R at nuclear or perinuclear locations also supports the view that IP R activation may be involved in activation of Orai and TRPC (at a putative lower level), as previously shown in other cell types [6,6]. The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 9

13 Ca channels in chicken sperm functions T.M.D. Nguyen et al. P.AMPKα (Thr7) (6 kda) AMPKα (TT) (6 kda) p.ampk/ampk mm Ca µm Nifedipine 60 nm SNX-8 0 µm MRS-85 b a Fig.. Effect of Ca channel inhibitors (nifedipine, SNX 8 and MRS 85) on AMPK phosphorylation in sperm. Sperm were incubated with 0 lm nifedipine, 60 nm SNX 8 or 0 lm MRS 85 or a mixture of the three inhibitors for 5 min in the presence of 5 mm Ca. The positive control comprised sperm incubated with 5 mm Ca and no Ca channel inhibitor. The negative control comprised sperm incubated in the absence of 5 mm Ca and no Ca channel inhibitor. The experiment was repeated four times; values (%) are means SEM. Different letters above bars indicate values that were statistically significantly different at P < In addition, the present work shows that extracellular Ca influx is directly involved in stimulation of AMPK phosphorylation in chicken spermatozoa. Indeed, inhibition of Ca influx by various Ca channel inhibitors decreased AMPK phosphorylation, and the SOC inhibitor MRS 85 was the most efficient, as it abolished AMPK phosphorylation in response to higher calcium-induced AMPK activation. AMPK is an evolutionarily conserved serine/threonine kinase that acts as a regulator of energy balance at both cellular and whole-body levels [6]. AMPK responds to an increase in AMP levels by increasing ab + ab + b + b ATP-generating pathways and reducing ATP-consuming metabolic pathways [6]. We have previously demonstrated that AMPK protein is highly expressed in chicken sperm, and regulates the most important functions of sperm: motility and AR [7]. Moreover, in the presence of extracellular Ca, AMPK phosphorylation was shown to occur mainly through the calcium/calmodulin-dependent protein kinase kinasemediated pathway [5], during which it is bound to Ca /CaM complexes [65]. Interestingly, TRPC and Orai were found to be localized in the same subcellular areas as the AMPK and calcium/calmodulin-dependent protein kinase kinases [7,5], i.e. the acrosome, midpiece and flagellum. Taken together, these results suggest that calcium signaling is involved in the regulation of energetic metabolism through AMPK as well as in the AR and motility. This signaling may involve calcium channels localized in the plasma membrane associated with the AMPK protein kinase (Fig. ). This result provides new insight regarding signaling pathways leading to AMPK activation in chicken spermatozoa in vitro, and suggests that the Ca influx through VOCCs is not sufficient to increase AMPK activation, and that strong SOC activation is necessary for rapid calcium entry leading to AMPK activation. In conclusion, our findings emphasize the crucial participation of the SOCs and HVA VOCCs in triggering sperm motility and the AR, providing the first step in birds toward revealing the molecular mechanism of this process, which appears to be essential for fertilization. Although the external signals that modulate sperm motility and the AR are not well understood, our observations are consistent with the known fundamental role of Ca in motility and the AR. However, the sperm Ca channels that affect sperm functions differ from those in many other animal classes, probably because of the complex oviparous internal fertilization system specific to birds. Table. Gene database annotations for VOCC and SOC candidates and IP R in chicken and mammalian orthologs. The presence of the various candidates in the gene annotation databases Ensembl and NCBI and mammalian orthologs is indicated. Channel types Channels Coding gene Chicken gene (Ensembl/NCBI ID) Mammalian ortholog VOCCs Ca v. CACNAD ENSGALG /ID95895 CACNAD Ca v. CACNAE ENSGALG /ID CACNAE Ca v. CACNAG ENSGALG /ID76985 CACNAG Ca v. CACNAH ENSGALG /ID656 CACNAH Ca v. CACNAI ENSGALG000000/ID7900 CACNAI SOC candidates Orai Orai ENSGALG /ID685 Orai TRPC TRPC ENSGALG /ID776 TRPC Other IP R ITPR ENSGALG /ID95 ITPR 9 The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies

14 T.M.D. Nguyen et al. Ca channels in chicken sperm functions Table. Similarity and homology of proteins in chicken and mammals (human/mouse/rat). The protein similarities were determined by bioinformatic analysis using the GENOMICUS version.0 PHYLOVIEW web tool ( search.pl). No similarity with rat was observed for Ca v.. Determination of sequence homology and identification of conserved domains were performed using the National Center for Biotechnology Information gene annotation database ( Channels Protein similarity versus mammals (%) Conserved domains Protein sequence homology (all sequences) (%) Target peptide homology (%) Ca v. H9/M9/R9 Voltage-gated calcium channel IQ H86/M86/R86 R67 Four ion transport protein domains Ca v. H86/M85/R8 Voltage-gated calcium channel IQ H85/M8/R8 R87 Four ion transport protein domains Ca v. H8/M80/R80 Ion transport protein H70/M70/R69 H50 Ca v. H78/M78/R79 Four ion transport protein domains H77/M78/R78 H50 Ca v. H86/M86 Four ion transport protein domains H79/M76/R77 H8 Orai H9/M9/R9 Mediator of CRAC channel activity H87/M8/R8 H89 Orai superfamily domain TRPC H98/M98/R98 Transient receptor ion channel II H9/M9/R88 H97 Ankyrin repeats IP R H97/M97/R96 Ion transport protein MIR domain Two RyR and IP R homology domains IP /RyR H9/M9/R9 R95 IQ, isoleucine glutamine motif; CRAC, calcium-receptor release-activated-channel; MIR (protein mannosyltransferase, IP R and RyR) domain; RyR, Ryanodine receptor; H, human; M, mouse; R, rat. MRS-85, -APB, YM-588 Ca Nifedipine, SNX-8 Fig.. Scheme for regulation of AMPK activity with respect to avian sperm functions. This hypothetical scheme shows the calcium signaling pathway involved in regulation of AMPK activation in chicken sperm. It shows the potential implications of entry of external Ca on calcium/calmodulin-dependent protein kinase kinase (CaMKK)-promoted activation of AMPK in chicken sperm. Solid green arrows illustrate established relationships between stimuli, signals, improved metabolic status and control of avian sperm functions. Dashed red arrows are used for Ca channel activity inhibition. The large green arrow indicates AMPK phosphorylation. Extracellular Intracellular AMPK Orai, TRPC S O C CaMKK ( ) AMPK V O C C Ca CaM P Ca v. Ca v. Involved in sperm motility and AR Ca Sperm membrane Experimental procedures Chemicals and reagents All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise noted. complete TM Mini EDTA-free protease inhibitor cocktail tablets were obtained from Roche Diagnostics (Mannheim, Germany). Tris/glycine buffer (09), Tris/glycine/SDS buffer (09) and Precision Plus Protein TM All Blue standards (catalog number 6 07) were obtained from Bio Rad (Hercules, CA, USA). Nifedipine (,-Dihydro-,6-dimethyl--(-nitrophe- The FEBS Journal 8 (06) ª 06 Federation of European Biochemical Societies 95