The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa

1 The Opioid Peptide Beta-endorphin Stimulates Acrosome Reaction in Human 1 Spermatozoa. 2 3 Running title: Opioids system and male fertility 4 Summary sentence: A novel role of the opioid system is demonstrated in the regulation of 5 sperm function; by showing that beta-endorphin is involved in acrosome reaction of human 6 sperm cells. 7 Keywords: opioid peptides, sperm, signalling pathways, acrosome reaction, male 8 fertility 9 10 Authors and Affiliations 11 Itziar Urizar,1 Haizea Estomba,1 Iraia Muñoa,1 Roberto Matorras,3 Antonia Esposito,3 Luz 12 Candenas,2 Francisco M. Pinto,2 Asier Valdivia,4 Jon Irazusta,1 and Nerea Subirán,1* 13 1 Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque 14 Country (UPV/EHU), Leioa, Bizkaia, Spain. 15 3 Human Reproduction Unit, Cruces Hospital, BioCruces, University of the Basque Country 16 2 Biological Chemistry Chemical Research Institute – CSIC / University of Seville. Seville, 17 Spain. Seville, Spain 18 4 Department of Cellular Biology and Histology, Faculty of Pharmacy, University of the 19 Basque Country(UPN/EHU), Vitoria-Gasteiz, Alava, Spain 20 21 Correspondence 22 Nerea Subirán, Department of Physiology, Faculty of Medicine and Dentistry, University of the 23 Basque Country 24 E-mail: nerea.sub[email protected] 25 Telephone: +34 94 601 5673 26 Fax: +34 94 601 5662, 27 28 2 ABSTRACT 29 The acrosome reaction occurs in vivo following sperm capacitation and is essential for 30 the acquisition of sperm fertilization ability. However, little is known about the 31 molecular identity of the physiological acrosome reaction regulators. In addition to 32 progesterone, which is produced by cumulus oophorus cells and known to regulate 33 acrosome reaction by activating the specific calcium channel CatSper, endogenous 34 opioid peptides such as beta-endorphin and met-enkephalin are present at high 35 concentrations in the follicular fluid suggesting that the opioid system may be involved 36 in the mechanisms regulating the acrosome reaction in humans. By using Reverse 37 Transcription-PCR, western blot and immunofluorescence approaches, we described the 38 presence and localization of the beta-endorphin precursor, pro-opiomelanocortin in the 39 middle section and in flagellum of human spermatozoa, and inside the seminiferous 40 tubules of human testis. Flow cytometry and intracellular calcium analyses showed that 41 beta-endorphin causes an inversely dose-dependent increase of the percentage of 42 acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. 43 These findings are important for future studies of sperm physiology and provide new 44 insight into the function of the opioid system as a target of fertility management. 45 46 47 3 INTRODUCTION 48 After ejaculation, human sperm cells are immature and infertile and must undergo many 49 modifications to become fertilization competent (Suarez 2008). Several morphological 50 and biochemical changes occur during the transit through the female tract. These sperm 51 modifications include different processes such as capacitation (sperm membrane 52 reorganization), hyperactive motility (changes to the motility pattern needed to 53 penetrate oocyte vestments) and acrosome reaction. The acrosome reaction of 54 spermatozoa is a complex calcium dependent process and is essential for the 55 spermatozoa to fertilize an egg. Fusion at multiple sites between the outer acrosomal 56 membrane and the cell membrane causes the release of the acrosomal contents and the 57 loss of the membranes surrounding the acrosome (Florman et al. 2008). 58 Progesterone produced by cumulus oophorus cells is known to be the main 59 physiological regulator of acrosome reaction (Baldi et al. 2009) since the binding and 60 the respond of progesterone are compromised in spermatozoa derived from infertile 61 men (Gadkar et al. 2002; Smith JF et al., 2013). Pregesterone-induced acrosome reaction 62 causes a multicomponent intracellular Ca2+ increases (Darszon et al. 2011). In 63 mammalian sperm, the progesterone-induced intracellular Ca2+ increase is controlled by 64 a sperm-specific Ca2+ channel called CatSper (cation channel of sperm) (Tamburrino L 65 et al., 2014; Quill et al., 2001; Lishko et al., 2010). However, several chemical 66 molecules including vitamin D, chemokines, small peptides, the gas NO, 67 neurotransmitters, analogues of cyclic nucleotides, and odorants can also affect 68 acrosomal exocytosis in vitro (Eisenbach and Giojalas, 2006; Florman et al., 2008; 69 Brenker et al., 2008; Suarez, 2008). To date, the underlying signalling mechanisms of 70 acrosome reaction are ill-defined. 71 72 4 Endogenous opioid peptides (EOPs) are a type of small peptides known to participate in 73 the regulation of reproductive physiology at multiple sites and, particularly, the opioid 74 system seems to be involved in the regulation of sperm physiology (Subirán et al. 75 2011). Previously, we described the presence of three types of opioid receptors (mu, 76 delta, kappa) and other components of the opioid system in human sperm cells and we 77 described its role in sperm motility. (Fernandez et al. 2002, Agirregoitia et al. 2006, 78 Subiran et al. 2008, 2012). Nevertheless, the role of the opioid system in acrosome 79 reaction is poorly understood and to date, there have been no relevant in vivo studies. - 80 endorphin immunoreactivity has been detected in spermatozoa but the main role of this 81 peptide in human spermatozoa is completely unknown. Together with progesterone, 82 beta-endorphin is secreted in the oviduct (Petraglia et al. 1986, 1986), raising the 83 possibility that EOPs may be involved in human acrosome reaction regulation. Here, we 84 describe for the first time that the EOP beta-endorphin precursor, pro-opiomelanocortin 85 (POMC), is present in human testis and sperm cells and that beta-endorphin regulates 86 human acrosome reaction by specific calcium -independent protein kinase C (PKC) 87 pathway. 88 89 MATERIALS AND METHODS 90 Samples and Isolation of Spermatozoa 91 Ethical approval for this study was obtained from the Ethics Committee of the 92 University of the Basque Country (CEISH/61/2011). Freshly ejaculated semen was 93 collected from 80 donors (18–35 years old) with normal sperm parameters according to 94 World Health Organization standards (WHO, 2010). Samples were obtained by 95 masturbation after 3–4 days of sexual abstinence and processed immediately upon 96 liquefaction (at 37ºC for 30 min). Spermatozoa were capacitated by a swim-up 97 5 procedure (Cejudo-Roman et al., 2013) and resuspended in G-IVF (Vitrolife, Göteborg, 98 Sweden) supplemented with 1% bovine serum albumin (BSA) for 3 h at 37°C under 5% 99 CO2. 100 101 Reverse Transcription-PCR (RT-PCR) Analysis 102 Total RNA was extracted from a sperm pool containing sperm from eight different 103 donors using TriReagent (Sigma, San Luis, MO, Estados Unidos) and cDNA was 104 synthesized using the Quantitect Reverse Transcription kit (Qiagen, Venlo, The 105 Netherlands). Specific oligonucleotide primer pairs used for PCR were synthesized and 106 purified by Sigma Genosys (Cambridge, UK) and their sequences were as follows: 107 human Pomc, forward 5ʹ-CTCACCACGGAAAGCAACC -3ʹ and reverse 5ʹ108 ATCGGTCCCAGCGGAAGT -3ʹ (151-bp product); and human Actb (-actin), forward 109 5ʹ-TCCCTGGAGAAGAGCTACGA-3ʹ and reverse 5ʹ110 ATCTGCTGGAAGGTGGACAG-3ʹ (362-bp product; exon spanning), used as an 111 internal control. 112 A pool of cDNAs from 20 different human tissues (human total RNA master panel, BD 113 Biosciences, Clontech, Palo Alto, CA, USA) was used as a positive control of 114 amplification. Amplification was carried out in 25 μl of PCR buffer containing 3 μl of 115 cDNA reaction mixture, 2.5 mM MgCl2, 0.2 μM primers, 200 μM dNTPs and 1.5 U of 116 heat-activated thermostable DNA polymerase (Immolase, Bioline, London, UK). PCR 117 was performed for 35 cycles with cycling parameters being: 15 s at 94°C, 20 s at 60°C 118 and 20 s at 72°C. The primers for hPomc were located on the same exon of each 119 respective gene (i.e. they did not span introns). Thus, we verified the possible carryover 120 of genomic DNA during the extraction process by performing PCR in the absence of 121 reverse transcriptase. Expression of CD4 and acrosin was also analysed to exclude the 122 6 presence of leukocyte contamination and to verify the presence of sperm 123 complementary DNA, respectively (data no shown). The RT-PCR products were 124 separated by 2.5% gel electrophoresis. The amplicon sizes were verified by comparison 125 with a DNA size-ladder and the identity of the products was established by sequencing 126 of amplicons. 127 128 Western blotting 129 Sperm proteins were prepared as described elsewhere (Subiran et al., 2012), modifying 130 the lysis buffer (phosphate-buffered saline [PBS] and 1% [v/v] Triton-X100, with 131 protease inhibitor cocktail). Membrane pellets were suspended in lysis buffer, and then 132 protein extracts were diluted in Laemmly sample buffer containing β-mercaptoethanol 133 (5% vol/vol) and boiled for 5 min. Proteins (50 µg sperm protein; 30 µg human 134 kidney´s cells´s protein) were loaded onto 12% resolving gels and separated by one135 dimensional SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride 136 membranes using the Mini Trans-Blot electrophoretic transfer system (Bio-Rad 137 Laboratories, Hercules, CA). After transfer, the membrane was blocked with Blotto (20 138 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% Triton X-100) containing 5% nonfat dry milk 139 (blocking buffer) for 1 h and then incubated with a dilution of polyclonal rabbit anti140 POMC antibody (1:200) After washing (3 x 5 min) in Blotto buffer, the membrane was 141 incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG antibody (1:3000) 142 (Goat anti-rabbit IgG HRP, abcam, ab6112). Blots were revealed for peroxidase activity 143 by enhanced chemiluminiscence (ECL). 144 145 Indirect Immunofluorescence 146 7 Isolated spermatozoa obtained after swim-up and human frozen testis slides provided by 147 Ziagen Company (Maryland, USA) were used to identify the localization of POMC in 148 human sperm cells and testis, and to analyze the effect of beta-endorphin on PKC149 signalling pathways. 150 Cells were fixed in 4% paraformaldehyde for 10 min, permeated in 0.5% Triton X-100 151 for 10 min and blocked for 30 min with 10% (v/v) fetal bovine serum in PBS. For 152 immunofluorescence staining, samples were incubated overnight at 4ºC with different 153 primary antibodies. We used rabbit polyclonal anti-proopiomelanocortin (1:500) (Santa 154 Cruz Technologies, California, USA) and rabbit anti-phospho-PKC (Cell Signalling) 155 antiserum at a dilution of 1:500. Secondary antibody incubations involved Alexa Fluor 156 488 donkey anti-rabbit IgG (1:2000) (Molecular Probes, Oregon USA). Nuclei were 157 stained with Hoechst 33342 at 10μg/ml (spermatozoa) and propidium iodide at 158 100g/ml (testis), and slides were assembled with Fluoromount G (Molecular Probes). 159 The specificity of the primary antibody was verified by using negative unspecific rabbit 160 immunoglobulin fraction (normal) (Dako) in the same concentration as the primary 161 antibody, and pre-absorbing primary antibody immunoreactivity with beta-endorphin 162 (10-5M) for 2 h at room temperature before incubation. At the same time, controls for 163 the specificity of the secondary antisera were performed by omitting the primary 164 antiserum before addition of the secondary antisera. Finally, the samples were examined 165 using confocal microscopy (Olympus Fluoview FV500, Tokyo, Japan). 166 Corrected total cell fluorescence (CTCF) per area was measured by ImageJ software 167 using the following equation: CTCF = [Integrated density – (Area of selected cell × 168 Mean fluorescence of background readings)] / Area of selected cell. We measured the 169 green fluorescence of at least 200 cells. 170 171 8 Incubation Media and Treatments 172 Isolated spermatozoa were treated at 37°C under 5% CO2 with different doses of beta173 endorphin (10–5, 10–7 and 10–9 M). Sperm samples were divided into aliquots of 0.1 ml 174 in G-IVF (Vitrolife) and one of the different concentrations of beta-endorphin was 175 added to each aliquot. An equal volume of solvent was used as control. In addition, to 176 ascertain the specificity of the action of the peptide, beta-endorphin (10–9 M)-treated 177 sperm cells and control samples were also co-incubated with naloxone, an antagonist of 178 opioid receptors, at high (10–5 M) and low (10–8 M) concentrations. High naloxone doses 179 (10-5 M) block the three opioid receptors and low doses (10-8 M) are able to block 180 selectively the mu-opioid receptor. Sperm cells were treated with naloxone for 10 min 181 before beta-endorphin addition. In all experiments, sperm cells were incubated with 182 beta-endorphin for 60 min. 183 Finally, to evaluate the effect of beta-endorphin on progesterone-induced acrosome 184 reaction, samples were also co-incubated with 10–9 M beta-endorphin and 10–6 M 185 progesterone. After 1 h of incubation with beta-endorphin, samples were treated with 186 progesterone for 15 min. 187 Treated spermatozoa were used for subsequent experiments. 188 189 Flow cytometry 190 In all experiments, acrosome reaction was measured by flow cytometry. We used 191 Fluorescein IsoTioCyanate (FITC) anti-human CD46 (for 60 min at room temperature; 192 BioLegend, California, USA) and Hoechst 33258 (2 min at room temperature; Sigma193 Aldrich, Missouri, USA) as acrosome reaction molecular marker and viability dyes, 194 respectively. Samples were checked visually by confocal microscopy to verify the 195 signal of the dyes. Green positive cells represented acrosome-reacted spermatozoa. 196 9 Fluorescence data from at least 100,000 events was analysed in a flow cytometer 197 (FACScalibur, Becton Dickinson, San Jose, CA, USA). To ensure fluorescence data 198 were from live spermatozoa, the percentage of Hoechst 33258-positive events was 199 determined by subtraction of background fluorescence in each histogram. 200 We also analysed the effect of beta-endorphin on PKC-signalling pathways using flow 201 cytometry. Capacitated spermatozoa obtained by Percoll gradient followed by a swim– 202 up procedure were incubated for 3 h in GVI-F® medium. A minimum of 3 x106 cell/ml 203 was collected and treated aswe described before. Collected spermatozoa were fixed and 204 permeated in suspension in 0.5% Triton X-100 for 10 min. Samples were washed twice 205 in PBS by centrifugation at 800 g for 5 min and incubated in blocking medium (PBS / 206 10% (v/v) fetal bovine serum) for 30 min. For immunofluorescence staining, samples 207 were incubated overnight at 4ºC with rabbit anti-phospho-PKC (Cell Signalling) 208 antiserum at a dilution of 1:500. On the next day, the samples were centrifuged in PBS 209 at 800 g for 5 min and incubated with Alexa Fluor 488 donkey anti-rabbit IgG (1:2000) 210 (Molecular Probes, Oregon USA) in the dark, at room temperature for 1 h . Nucleus 211 was stained with 0.1 μg/ml Hoechst 33258 for 2 min. Finally, samples were washed 212 twice by centrifugation in PBS at 800 g for 5 min, suspended in PBS and kept in the 213 dark until analysis. Negative controls were performed by omitting the primary antibody 214 before secondary antibody addition and by using negative unspecific rabbit 215 immunoglobulin fraction (normal) (Dako) in the same concentration as the primary 216 antibody. Fluorescence data from at least 100,000 events were analyzed. In order to 217 measure the green fluorescence only from spermatozoa, the percentage of Hoechst 218 33258-positive was determined by subtraction of background fluorescence in each 219 histogram. Histograms were analyzed using the Summit v4.3 software. 220 221 16 concentration of the cytosol that is required for the acrosomic reaction (Ickowicz et al. 372 2012; Vigil et al. 2011). 373 Our results suggest that the opioid peptide beta-endorphin can be a physiological 374 inductor of the acrosome reaction. We found an inverse dose-dependent activation of 375 acrosome reaction induced by beta-endorphin. In fact, the physiological doses of beta376 endorphin (10–9 M) caused the most potent effect on acrosome reaction. High doses (10– 377 5 M) of the specific antagonist, naloxone, blunted the activation of acrosome reaction, 378 suggesting that the effect of beta-endorphin is specifically mediated by activation of the 379 opioid receptors. However, low doses of naloxone (10–8 M) -at which this compound 380 acts selectively on the mu-opioid receptoronly partially blocked the effect of beta381 endorphin on acrosome-reacted spermatozoa, raising the possibility that more than one 382 receptor might be involved in this process The activation of more than one type of 383 opioid receptor also can explain the inverse dose-dependent inhibition, since the mu384 and delta-opioid receptors can activate opposite responses, as we observed in human 385 sperm motility (Agirregoitia et al. 2006). 386 Together with progesterone, beta-endorphin is present at high concentrations in the 387 follicular fluid and in the vicinity of the egg (Petraglia et al. 1985, 1986). To elucidate 388 whether beta-endorphin modulates progesterone action, we co-incubated sperm cells 389 with both beta-endorphin and progesterone. Beta-endorphin modified the progesterone390 response. The percentage of acrosome-reacted sperm cells in samples co-incubated with 391 beta-endorphin and progesterone was 1.5-fold higher that in samples with only 392 progesterone. 393 Owing to the fact that the progesterone response is totally dependent on Ca2+/PKC 394 pathways (O'Toole et al. 1996; Chen et al., 2000; Rathi et al., 2003), we investigated 395 whether beta-endorphin may stimulates acrosome reaction by activation of the 396 17 Ca2+/PKC pathway. A common and fundamental feature of physiological and 397 pharmacological acrosome reaction inducers is that they provoke intracellular 398 multicomponent Ca2+ increases (Darszon et al. 2011). Thus, we investigated whether 399 beta-endorphin stimulates an increase in intracellular Ca2+. Progesterone caused a 400 typical biphasic wave of intracellular Ca2+ stimulation in sperm, composed of a transient 401 increase followed by a sustained elevation as previously reported (Baldi et al. 2009, 402 Gadkar et al. 2002). We failed to detect any change in Ca2+ after addition of beta403 endorphin. Beta-endorphin did not cause any effect on spermatozoa [Ca2+]i in Fura-2404 loaded sperm suspensions and none of the doses assayed was able to modify the 405 progesterone-induced calcium response. In spite of that, beta-endorphin caused an 406 activation of the PKC-induced substrates phosphorylation. By immunofluorescence and 407 flow cytometry approaches, we observed an increase in the phosphorylation of PKC408 induced substrates after beta-endorphin exposure. In addition, we also reported a further 409 activation of the PKC-signalling pathway in semen samples co-incubated 410 simultaneously with beta-endorphin and progesterone. Compared to progesterone alone, 411 the co incubation of beta-endorphine and progesterone caused a 1.7-fold and 1.2-fold 412 increase in the phosphorylation of PKC substrates, measured by CTCF and flow 413 cytometry respectively. This result was also consistent with the increase observed in the 414 percentage of acrosome reacted sperm cells. Thus, beta-endorphin may stimulate the 415 acrosome reaction via PKC-signalling pathway activation, as have been reported for 416 other inductors (Vigil et al. 2011, O'Toole et al. 1996). Moreover, our data suggest that 417 beta-endorphin can activate the PKC-signalling pathways through a Ca2+-independent 418 pathway. Mouse and rat eggs can express the atypical Ca2+-independent PKC isoforms ζ 419 and λ, (Pauken et al. 2000, Page et al. 2004) but further analyses will be necessary to 420 analyze the presence of Ca2+-independent PKC isoforms in human sperm cells. 421 18 In conclusion, the present data allow us to identify a new physiological acrosome422 reaction inductor and described its signalling pathways in human sperm. Beta-endorphin 423 may be involved in the regulation of acrosome reaction by a Ca2+-independent PKC 424 pathway in humans. These findings are important for future studies of sperm physiology 425 and provide new insight into the function of the opioid system as a target for fertility 426 management. 427 428 19 Acknowledgements 429 This work was supported by grants from The Basque Government and University of the 430 Basque Country (UPV/EHU) and Ministerio de Economía y Competitividad 431 (CTQ2011-25564). HE was supported by fellowship from Jesus Gangoiti Barrera 432 Foundation. IM was supported by fellowship from Basque Government. IU was 433 supported by fellowship from University of Basque Country (UPV/EHU). 434 435 Disclosures 436 The authors have nothing to disclose 437 438 Author's contributions 439 I.U., H.E., and I.M carried out and analyzed the experiments, F.M.P. and L.C. carried 440 out the experiments and provided conceptual support, R.M and A.E evaluated the 441 samples., A.V and J.I. provided conceptual support N.S. designed the study, analyzed 442 the experiments and wrote the manuscript. 443 444 20 REFERENCES 445  Agirregoitia E, Carracedo A, Subirán N, Valdivia A, Agirregoitia N, Peralta L, 446 Velasco G, Irazusta J. (2010) The CB(2) cannabinoid receptor regulates human 447 sperm cell motility. Fertil Steril; Mar 15;93(5):1378-87 448  Agirregoitia E, Valdivia A, Carracedo A, Casis L, Gil J, Subiran N, Ochoa C, 449 Irazusta J. (2006) Expression and localization of delta-, kappa-, and mu-opioid 450 receptors in human spermatozoa and implications for sperm motility. 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A) RT-PCR analysis of 553 proopiomelanocortin (POMC) precursor in human spermatozoa (sp1 and sp2); nc: primers 554 without cDNA were used as negative control and pc: pool of DNA from 20 different human 555 tissues used as a positive control. B) Western blotting analysis of POMC in human spermatozoa 556 (Sp) and kidney (kd) using a rabbit anti-POMC polyclonal antiserum. The molecular mass 557 markers (kDa) are indicated on the left. Molecular weights of pre-stained markers proteins are 558 indicated. Representative blot obtained from four normozoospermic donors is shown C) 559 Immunofluorescence analysis of POMC in human sperm cells (panel 1). Negative controls 560 incubating with unspecific rabbit immunoglobulin fraction (panel 2) and preadsorbing the anti561 POMC antibody with beta-endorphin (panel 3). Incubation with secondary antibody alone 562 (panels 4). DNA of controls was stained with Hoechst 33342. Representative photomicrographs 563 are shown; n = 5. Scale bar for all panels, 1 D) Immunofluorescence analysis of POMC in 564 human testis (panel 1). Negative controls incubating with unspecific rabbit immunoglobulin 565 fraction (panel 2) and preadsorbing the anti-POMC antibody with beta-endorphin (panel 3). 566 Incubation with secondary antibody alone (panels 4). DNA of controls was stained with 567 Propidium Iodide. Representative photomicrographs are shown; n = 3. Scale bar for all panels, 568 50 µm. 569 570 FIG. 2. Effect of beta-endorphin on human acrosome reaction. A) Dose-dependent effect of 571 beta-endorphin on the percentage of acrosome-reacted sperm cells for 1 h. B) Percentage of 572 CD46-positive sperm cells after co-incubation with beta-endorphin (10–9 M) and high (10–5 M) 573 and low doses (10–8 M) of naloxone for 1 h. C) Percentage of CD46-positive sperm cells after 574 co-incubation with beta-endorphin (10–9 M) and progesterone (10–6 M) for 1 h. * P < 0.05, 575 significant difference vs control responses; ** P < 0.01, significant difference vs control 576 25 responses; and + P < 0.05 significant difference vs beta-endorphin responses. (n = 12). 577 Normalized data as [(Treatment – Control)/(Control)] × 100 578 579 FIG.3. Effects of beta-endorphin on intracellular free Ca2+ ([Ca 2+ ] i ). A) 580 Intracellular free Ca2+ measurement in human sperm cells loaded with Fura-2 in response 581 to beta-endorphin (10-9 M) (red line) and control (black line). Subsequent addition of 10–6 M 582 progesterone to the same sperm aliquot caused a typical biphasic [Ca2+]i progesterone response 583 that had not been modified by beta-endorphin. The X axis shows time in seconds and the Y axis 584 shows [Ca2+]i data expressed by the F340/F380 ratio. TX= Triton X-100. Traces are 585 representative of typical results obtained in five different experiments for each blocker. B) 586 Dose-dependent effect of beta-endorphin on progesterone-induced intracellular Ca2+ response. 587 Data expressed the area of the progesterone-induced [Ca2+]i signal measured by the ratio of 588 F340/F380 signals. Calibration of [Ca2+]i was achieved adding Triton X-100 (TX), to obtain 589 the maximal response, followed by addition of EGTA to obtain the minimal response. n=5. 590 591 FIG. 4. Effect of beta-endorphin on Ca2+/protein kinas C (PKC)-signalling pathway. A) 592 Immunofluorescence analysis of the PKC-induced substrate phosphorylation in samples treated 593 with beta-endorphin, beta-endorphin and progesterone, and progesterone. DNA of controls was 594 stained with Hoechst 33342. Representative photomicrographs are shown; n = 5. Scale bar, 2 595 μm. B) Percentage of phospho-PKC substrates positive spermatozoa and C) fluorescence 596 intensity measured by flow cytometry in samples treated with beta-endorphin, beta-endorphin 597 and progesterone, and progesterone. Fluorescence data from at least 100,000 events was 598 analyzed. *p < 0.05, significant difference vs control responses; **p < 0.01, significant 599 difference vs control responses; and + p < 0.01, significant difference vs progesterone 600 responses. 601