76a1112b8641c12fffafae0f5535027e 2022_Castro-Arnau_et_al_IJMS.pdf b4a5e175d21365bf6c75522758fb98f336501d09 2022_Castro-Arnau_et_al_IJMS.pdf 8185932c830c4b51c0a3bda09ae765fafd0085516fe5f766da118d3832a9ce97 2022_Castro-Arnau_et_al_IJMS.pdf Title: Role of Ion Channels in the Maintenance of Sperm Motility and Swimming Behavior in a Marine Teleost Subject: In oviparous marine fishes, the hyperosmotic induction of sperm motility in seawater (SW) is well established, however, the potential function of ion channels in the maintenance of post activated spermatozoon swimming performance remains largely unknown. Here, we investigated the influence of ion channels on the spermatozoon swimming parameters using the gilthead seabream (Sparus aurata) as a model for modern marine teleosts. Our data show that the SW-induced activation of seabream sperm motility requires three concomitant processes, the hyperosmotic shock, an ion-flux independent increase of the intracellular concentration of Ca2+ ([Ca2+]i), but not of [K+]i or [Na+]i, and the alkalization of the cytosol. The combination of all three processes is obligatory to trigger flagellar beating. However, the time-course monitoring of sperm motion kinetics and changes in the [Ca2+]i, [K+]i and [Na+]i in SW or in non-ionic activation media, showed that the post activated maintenance of spermatozoa motility is dependent on extracellular Ca2+ and K+. A meta-analysis of a seabream sperm transcriptome uncovered the expression of multiple ion channels, some of which were immunolocalized in the head and/or tail of the spermatozoon. Selective pharmacological inhibition of these ion channel families impaired the long-term motility, progressivity, and velocity of SW-activated spermatozoa. The data further revealed that some antagonists of K+-selective or Ca2+-selective channels, as well as of stretch-activated and mechanosensitive channels, altered the trajectory of spermatozoa, suggesting that these ion channels are likely involved in the control of the swimming pattern of the post activated spermatozoon. These combined findings provide new insight into the signaling pathways regulating spermatozoon activation and swimming performance in marine fishes. Keywords: spermatozoa; ions; pH; activation; transcriptome; ion channels; motility; trajectory Author: Júlia Castro-Arnau, François Chauvigné and Joan Cerdà Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Oct 13 08:11:44 2022 CEST ModDate: Thu Oct 13 08:24:01 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 7122448 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SYYJVK+VnURWPalladioL Type 1 Custom yes yes no 10 0 JCYBOT+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 EQUCOC+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 OFCDUU+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 AALFUC+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 JDCKWC+CMSY10 Type 1 Builtin yes yes no 71 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 81 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-03-08 02:37:23 CET RepresentationInformation: 2022_Castro-Arnau_et_al_IJMS.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-03-06 01:34:44 CET Size: 7122448 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 487 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: A Surge of Intracellular Ca2+, but Not of K+ or Na+, Is Necessary for Sperm Motility Activation in the Seabream Destination: subsection.2.1 Item: Title: Basic pH Is Necessary to Activate and Maintain Sperm Motility Destination: subsection.2.2 Item: Title: Ca2+ and pHi Are Not Sufficient to Trigger Sperm Motility Destination: subsection.2.3 Item: Title: External Ca2+ and K+ Are Required for the Post Activation Maintenance of Sperm Motility Destination: subsection.2.4 Item: Title: Seabream Spermatozoa Express Multiple Ion Channels Destination: subsection.2.5 Item: Title: Ion Channel Blockers Affect the Motility of Post Activated Spermatozoa Destination: subsection.2.6 Item: Title: Ion Channels Can Control the Sperm Swimming Pattern Destination: subsection.2.7 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Animals and Semen Collection Destination: subsection.4.1 Item: Title: Reagents and Antibodies Destination: subsection.4.2 Item: Title: Sperm Motility Assays Destination: subsection.4.3 Item: Title: Measurement of Intracellular Ions Destination: subsection.4.4 Item: Title: Meta-Analysis of a Seabream Sperm Transcriptome Destination: subsection.4.5 Item: Title: RT-PCR Expression Analysis Destination: subsection.4.6 Item: Title: Biochemical Fractionation of Spermatozoa and Protein Extraction Destination: subsection.4.7 Children: Item: Title: Total Sperm Destination: subsubsection.4.7.1 Item: Title: Total Membrane Destination: subsubsection.4.7.2 Item: Title: Isolation of Flagella Destination: subsubsection.4.7.3 Item: Title: Immunoblotting Destination: subsection.4.8 Item: Title: Immunofluorescence Microscopy Destination: subsection.4.9 Item: Title: Statistical Analyses Destination: subsection.4.10 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Role of Ion Channels in the Maintenance of Sperm Motility and Swimming Behavior in a Marine Teleost Author: Júlia Castro-Arnau, François Chauvigné and Joan Cerdà Subject: In oviparous marine fishes, the hyperosmotic induction of sperm motility in seawater (SW) is well established, however, the potential function of ion channels in the maintenance of post activated spermatozoon swimming performance remains largely unknown. Here, we investigated the influence of ion channels on the spermatozoon swimming parameters using the gilthead seabream (Sparus aurata) as a model for modern marine teleosts. Our data show that the SW-induced activation of seabream sperm motility requires three concomitant processes, the hyperosmotic shock, an ion-flux independent increase of the intracellular concentration of Ca2+ ([Ca2+]i), but not of [K+]i or [Na+]i, and the alkalization of the cytosol. The combination of all three processes is obligatory to trigger flagellar beating. However, the time-course monitoring of sperm motion kinetics and changes in the [Ca2+]i, [K+]i and [Na+]i in SW or in non-ionic activation media, showed that the post activated maintenance of spermatozoa motility is dependent on extracellular Ca2+ and K+. A meta-analysis of a seabream sperm transcriptome uncovered the expression of multiple ion channels, some of which were immunolocalized in the head and/or tail of the spermatozoon. Selective pharmacological inhibition of these ion channel families impaired the long-term motility, progressivity, and velocity of SW-activated spermatozoa. The data further revealed that some antagonists of K+-selective or Ca2+-selective channels, as well as of stretch-activated and mechanosensitive channels, altered the trajectory of spermatozoa, suggesting that these ion channels are likely involved in the control of the swimming pattern of the post activated spermatozoon. These combined findings provide new insight into the signaling pathways regulating spermatozoon activation and swimming performance in marine fishes. Keywords: spermatozoa; ions; pH; activation; transcriptome; ion channels; motility; trajectory Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Oct 13 08:11:44 CEST 2022 ModDate: Thu Oct 13 08:24:01 CEST 2022 ID: 0x80add32badbdb5c3a39ffb9a9661fc72, 0x80add32badbdb5c3a39ffb9a9661fc72 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: EQUCOC+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: EQUCOC+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JDCKWC+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: JDCKWC+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: OFCDUU+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: OFCDUU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SYYJVK+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: SYYJVK+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: AALFUC+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: AALFUC+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JCYBOT+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 231 FontDescriptor: FontName: JCYBOT+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 80, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 126, 660, 137, 674 Annotation: Subtype: Link Rect: 323, 660, 335, 674 Annotation: Subtype: Link Rect: 206, 172, 213, 181 ActionDest: 171 Annotation: Subtype: Link Rect: 421, 147, 428, 156 ActionDest: 171 Annotation: Subtype: Link Rect: 269, 109, 276, 119 ActionDest: 171 Annotation: Subtype: Link Rect: 277, 110, 284, 119 ActionDest: 171 Annotation: Subtype: Link Rect: 383, 72, 390, 81 ActionDest: 171 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 408, 87, 427 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 405, 710, 412, 719 ActionDest: 171 Annotation: Subtype: Link Rect: 347, 635, 354, 644 ActionDest: 171 Annotation: Subtype: Link Rect: 355, 635, 362, 644 ActionDest: 171 Annotation: Subtype: Link Rect: 237, 597, 244, 606 ActionDest: 171 Annotation: Subtype: Link Rect: 274, 572, 281, 581 ActionDest: 171 Annotation: Subtype: Link Rect: 282, 572, 289, 581 ActionDest: 171 Annotation: Subtype: Link Rect: 549, 560, 556, 569 ActionDest: 171 Annotation: Subtype: Link Rect: 215, 522, 222, 531 ActionDest: 171 Annotation: Subtype: Link Rect: 544, 497, 556, 506 ActionDest: 171 Annotation: Subtype: Link Rect: 411, 447, 418, 456 ActionDest: 171 Annotation: Subtype: Link Rect: 355, 397, 362, 406 ActionDest: 171 Annotation: Subtype: Link Rect: 362, 396, 369, 406 ActionDest: 171 Annotation: Subtype: Link Rect: 370, 397, 382, 406 ActionDest: 171 Annotation: Subtype: Link Rect: 257, 372, 264, 380 ActionDest: 171 Annotation: Subtype: Link Rect: 401, 334, 408, 343 ActionDest: 171 Annotation: Subtype: Link Rect: 408, 334, 415, 343 ActionDest: 171 Annotation: Subtype: Link Rect: 416, 334, 423, 343 ActionDest: 171 Annotation: Subtype: Link Rect: 423, 334, 435, 343 ActionDest: 171 Annotation: Subtype: Link Rect: 536, 309, 543, 318 ActionDest: 171 Annotation: Subtype: Link Rect: 544, 309, 556, 318 ActionDest: 171 Annotation: Subtype: Link Rect: 207, 246, 214, 255 ActionDest: 171 Annotation: Subtype: Link Rect: 215, 246, 222, 255 ActionDest: 171 Annotation: Subtype: Link Rect: 222, 246, 234, 255 ActionDest: 171 Annotation: Subtype: Link Rect: 309, 196, 316, 205 ActionDest: 171 Annotation: Subtype: Link Rect: 316, 196, 328, 205 ActionDest: 171 Annotation: Subtype: Link Rect: 329, 196, 341, 205 ActionDest: 171 Annotation: Subtype: Link Rect: 347, 133, 359, 142 ActionDest: 171 Annotation: Subtype: Link Rect: 362, 133, 374, 142 ActionDest: 171 Annotation: Subtype: Link Rect: 536, 108, 543, 117 ActionDest: 171 Annotation: Subtype: Link Rect: 544, 108, 556, 117 ActionDest: 171 Annotation: Subtype: Link Rect: 473, 70, 485, 79 ActionDest: 171 Annotation: Subtype: Link Rect: 485, 70, 497, 79 ActionDest: 171 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 258, 735, 270, 744 ActionDest: 171 Annotation: Subtype: Link Rect: 319, 508, 331, 517 ActionDest: 171 Annotation: Subtype: Link Rect: 331, 508, 343, 517 ActionDest: 171 Annotation: Subtype: Link Rect: 263, 468, 270, 480 ActionDest: 75 Annotation: Subtype: Link Rect: 352, 405, 359, 417 ActionDest: 78 Annotation: Subtype: Link Rect: 264, 342, 271, 354 ActionDest: 78 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 459, 212, 466, 224 ActionDest: 78 Annotation: Subtype: Link Rect: 344, 187, 351, 199 ActionDest: 78 Annotation: Subtype: Link Rect: 334, 162, 341, 174 ActionDest: 78 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 234, 683, 241, 696 ActionDest: 78 Annotation: Subtype: Link Rect: 413, 610, 425, 619 ActionDest: 171 Annotation: Subtype: Link Rect: 426, 610, 438, 619 ActionDest: 174 Annotation: Subtype: Link Rect: 440, 610, 452, 619 ActionDest: 174 Annotation: Subtype: Link Rect: 185, 547, 197, 556 ActionDest: 171 Annotation: Subtype: Link Rect: 239, 519, 246, 531 ActionDest: 88 Annotation: Subtype: Link Rect: 237, 494, 243, 506 ActionDest: 88 Annotation: Subtype: Link Rect: 319, 110, 331, 119 ActionDest: 174 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 305, 733, 312, 746 ActionDest: 94 Annotation: Subtype: Link Rect: 544, 720, 551, 732 ActionDest: 94 Annotation: Subtype: Link Rect: 359, 272, 366, 284 ActionDest: 100 Annotation: Subtype: Link Rect: 395, 235, 402, 247 ActionDest: 100 Annotation: Subtype: Link Rect: 346, 184, 354, 197 ActionDest: 100 Annotation: Subtype: Link Rect: 200, 159, 207, 173 ActionDest: 100 Annotation: Subtype: Link Rect: 488, 134, 495, 146 ActionDest: 100 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 227, 289, 239, 298 ActionDest: 171 Annotation: Subtype: Link Rect: 239, 289, 251, 298 ActionDest: 171 Annotation: Subtype: Link Rect: 200, 273, 207, 285 ActionDest: 100 Annotation: Subtype: Link Rect: 296, 223, 303, 237 ActionDest: 100 Annotation: Subtype: Link Rect: 348, 198, 355, 212 ActionDest: 100 Annotation: Subtype: Link Rect: 405, 100, 417, 109 ActionDest: 174 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 547, 733, 554, 746 ActionDest: 106 Annotation: Subtype: Link Rect: 248, 197, 259, 205 ActionDest: 174 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 200, 683, 207, 695 ActionDest: 106 Annotation: Subtype: Link Rect: 200, 595, 207, 607 ActionDest: 106 Annotation: Subtype: Link Rect: 307, 557, 314, 569 ActionDest: 106 Annotation: Subtype: Link Rect: 409, 469, 416, 481 ActionDest: 115 Annotation: Subtype: Link Rect: 372, 444, 379, 456 ActionDest: 115 Annotation: Subtype: Link Rect: 358, 419, 364, 431 ActionDest: 115 Annotation: Subtype: Link Rect: 346, 381, 353, 393 ActionDest: 115 Annotation: Subtype: Link Rect: 416, 319, 423, 331 ActionDest: 115 Annotation: Subtype: Link Rect: 200, 293, 207, 305 ActionDest: 115 Annotation: Subtype: Link Rect: 478, 281, 485, 293 ActionDest: 115 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 499, 188, 506, 200 ActionDest: 121 Annotation: Subtype: Link Rect: 307, 125, 314, 137 ActionDest: 125 Annotation: Subtype: Link Rect: 333, 125, 340, 137 ActionDest: 131 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 199, 507, 206, 519 ActionDest: 125 Annotation: Subtype: Link Rect: 335, 470, 341, 482 ActionDest: 125 Annotation: Subtype: Link Rect: 419, 457, 426, 469 ActionDest: 125 Annotation: Subtype: Link Rect: 309, 382, 316, 394 ActionDest: 125 Annotation: Subtype: Link Rect: 430, 357, 437, 369 ActionDest: 125 Annotation: Subtype: Link Rect: 357, 332, 365, 344 ActionDest: 125 Annotation: Subtype: Link Rect: 251, 294, 258, 306 ActionDest: 125 Annotation: Subtype: Link Rect: 323, 246, 335, 256 ActionDest: 174 Annotation: Subtype: Link Rect: 200, 194, 207, 206 ActionDest: 125 Annotation: Subtype: Link Rect: 301, 169, 308, 181 ActionDest: 125 Annotation: Subtype: Link Rect: 488, 131, 495, 143 ActionDest: 131 Annotation: Subtype: Link Rect: 542, 118, 550, 130 ActionDest: 125 Annotation: Subtype: Link Rect: 245, 93, 252, 105 ActionDest: 131 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 200, 63, 207, 75 ActionDest: 131 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 353, 720, 360, 732 ActionDest: 131 Annotation: Subtype: Link Rect: 540, 708, 547, 720 ActionDest: 131 Annotation: Subtype: Link Rect: 436, 94, 443, 108 ActionDest: 131 Annotation: Subtype: Link Rect: 243, 57, 250, 69 ActionDest: 131 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 264, 720, 271, 732 ActionDest: 131 Annotation: Subtype: Link Rect: 231, 683, 238, 695 ActionDest: 131 Annotation: Subtype: Link Rect: 549, 582, 556, 594 ActionDest: 140 Annotation: Subtype: Link Rect: 268, 544, 275, 556 ActionDest: 140 Annotation: Subtype: Link Rect: 199, 519, 205, 531 ActionDest: 140 Annotation: Subtype: Link Rect: 498, 494, 505, 506 ActionDest: 140 Annotation: Subtype: Link Rect: 483, 469, 490, 481 ActionDest: 140 Annotation: Subtype: Link Rect: 374, 431, 381, 443 ActionDest: 140 Annotation: Subtype: Link Rect: 547, 406, 554, 418 ActionDest: 140 Annotation: Subtype: Link Rect: 353, 268, 365, 280 ActionDest: 146 Annotation: Subtype: Link Rect: 247, 243, 259, 257 ActionDest: 146 Annotation: Subtype: Link Rect: 302, 230, 314, 242 ActionDest: 146 Annotation: Subtype: Link Rect: 450, 218, 462, 230 ActionDest: 146 Annotation: Subtype: Link Rect: 365, 193, 377, 205 ActionDest: 146 Annotation: Subtype: Link Rect: 200, 180, 213, 192 ActionDest: 146 Annotation: Subtype: Link Rect: 341, 180, 353, 192 ActionDest: 146 Annotation: Subtype: Link Rect: 473, 180, 485, 192 ActionDest: 146 Annotation: Subtype: Link Rect: 199, 168, 211, 180 ActionDest: 146 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 343, 324, 349, 335 ActionDest: 125 Annotation: Subtype: Link Rect: 368, 324, 375, 335 ActionDest: 131 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 226, 282, 238, 291 ActionDest: 171 Annotation: Subtype: Link Rect: 238, 282, 250, 291 ActionDest: 171 Annotation: Subtype: Link Rect: 216, 131, 228, 140 ActionDest: 171 Annotation: Subtype: Link Rect: 228, 131, 240, 140 ActionDest: 171 Annotation: Subtype: Link Rect: 378, 81, 390, 90 ActionDest: 174 Annotation: Subtype: Link Rect: 393, 81, 405, 90 ActionDest: 174 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 350, 673, 362, 682 ActionDest: 174 Annotation: Subtype: Link Rect: 363, 673, 375, 682 ActionDest: 174 Annotation: Subtype: Link Rect: 375, 673, 387, 682 ActionDest: 174 Annotation: Subtype: Link Rect: 313, 647, 325, 657 ActionDest: 174 Annotation: Subtype: Link Rect: 436, 635, 448, 644 ActionDest: 171 Annotation: Subtype: Link Rect: 449, 635, 461, 644 ActionDest: 171 Annotation: Subtype: Link Rect: 274, 623, 286, 631 ActionDest: 171 Annotation: Subtype: Link Rect: 274, 585, 281, 594 ActionDest: 171 Annotation: Subtype: Link Rect: 282, 585, 294, 594 ActionDest: 174 Annotation: Subtype: Link Rect: 372, 547, 384, 556 ActionDest: 174 Annotation: Subtype: Link Rect: 385, 547, 397, 556 ActionDest: 174 Annotation: Subtype: Link Rect: 498, 535, 510, 544 ActionDest: 171 Annotation: Subtype: Link Rect: 511, 535, 523, 544 ActionDest: 174 Annotation: Subtype: Link Rect: 415, 509, 427, 519 ActionDest: 174 Annotation: Subtype: Link Rect: 468, 497, 475, 506 ActionDest: 171 Annotation: Subtype: Link Rect: 476, 497, 488, 506 ActionDest: 171 Annotation: Subtype: Link Rect: 488, 497, 500, 506 ActionDest: 174 Annotation: Subtype: Link Rect: 265, 472, 277, 481 ActionDest: 171 Annotation: Subtype: Link Rect: 277, 472, 289, 481 ActionDest: 174 Annotation: Subtype: Link Rect: 197, 422, 209, 431 ActionDest: 171 Annotation: Subtype: Link Rect: 210, 422, 222, 431 ActionDest: 174 Annotation: Subtype: Link Rect: 225, 422, 237, 431 ActionDest: 174 Annotation: Subtype: Link Rect: 427, 346, 439, 355 ActionDest: 174 Annotation: Subtype: Link Rect: 294, 321, 306, 330 ActionDest: 174 Annotation: Subtype: Link Rect: 305, 309, 317, 318 ActionDest: 174 Annotation: Subtype: Link Rect: 278, 233, 290, 242 ActionDest: 174 Annotation: Subtype: Link Rect: 417, 233, 429, 242 ActionDest: 174 Annotation: Subtype: Link Rect: 429, 233, 441, 242 ActionDest: 174 Annotation: Subtype: Link Rect: 199, 183, 210, 192 ActionDest: 174 Annotation: Subtype: Link Rect: 311, 145, 318, 154 ActionDest: 171 Annotation: Subtype: Link Rect: 319, 146, 331, 154 ActionDest: 174 Annotation: Subtype: Link Rect: 331, 146, 343, 154 ActionDest: 174 Annotation: Subtype: Link Rect: 344, 145, 356, 154 ActionDest: 174 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 403, 648, 410, 657 ActionDest: 171 Annotation: Subtype: Link Rect: 410, 647, 417, 657 ActionDest: 171 Annotation: Subtype: Link Rect: 417, 647, 429, 657 ActionDest: 171 Annotation: Subtype: Link Rect: 430, 648, 442, 657 ActionDest: 171 Annotation: Subtype: Link Rect: 442, 647, 454, 657 ActionDest: 174 Annotation: Subtype: Link Rect: 455, 648, 467, 657 ActionDest: 174 Annotation: Subtype: Link Rect: 467, 647, 479, 657 ActionDest: 174 Annotation: Subtype: Link Rect: 482, 647, 494, 657 ActionDest: 177 Annotation: Subtype: Link Rect: 228, 346, 240, 355 ActionDest: 177 Annotation: Subtype: Link Rect: 240, 346, 252, 355 ActionDest: 177 Annotation: Subtype: Link Rect: 544, 95, 556, 104 ActionDest: 174 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 463, 735, 470, 744 ActionDest: 171 Annotation: Subtype: Link Rect: 204, 609, 216, 618 ActionDest: 177 Annotation: Subtype: Link Rect: 433, 417, 440, 429 ActionDest: 75 Annotation: Subtype: Link Rect: 446, 420, 458, 429 ActionDest: 177 Annotation: Subtype: Link Rect: 424, 395, 436, 404 ActionDest: 177 Annotation: Subtype: Link Rect: 464, 379, 471, 391 ActionDest: 75 Annotation: Subtype: Link Rect: 410, 267, 417, 280 ActionDest: 75 Annotation: Subtype: Link Rect: 479, 181, 491, 190 ActionDest: 171 Annotation: Subtype: Link Rect: 491, 181, 503, 190 ActionDest: 177 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 306, 346, 318, 355 ActionDest: 174 Annotation: Subtype: Link Rect: 375, 318, 559, 330 Annotation: Subtype: Link Rect: 298, 308, 310, 317 ActionDest: 177 Annotation: Subtype: Link Rect: 369, 293, 494, 305 Annotation: Subtype: Link Rect: 227, 270, 239, 279 ActionDest: 177 Annotation: Subtype: Link Rect: 375, 258, 387, 267 ActionDest: 174 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 544, 543, 556, 552 ActionDest: 177 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 536, 294, 560, 305 Annotation: Subtype: Link Rect: 165, 282, 381, 293 Annotation: Subtype: Link Rect: 441, 112, 559, 123 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 330, 712, 341, 720 ActionDest: 174 Annotation: Subtype: Link Rect: 59, 607, 96, 617 Annotation: Subtype: Link Rect: 102, 607, 138, 617 Annotation: Subtype: Link Rect: 259, 583, 296, 594 Annotation: Subtype: Link Rect: 303, 583, 339, 594 Annotation: Subtype: Link Rect: 272, 560, 310, 571 Annotation: Subtype: Link Rect: 246, 537, 284, 548 Annotation: Subtype: Link Rect: 290, 537, 326, 548 Annotation: Subtype: Link Rect: 447, 514, 484, 527 Annotation: Subtype: Link Rect: 491, 514, 527, 527 Annotation: Subtype: Link Rect: 479, 503, 515, 514 Annotation: Subtype: Link Rect: 521, 503, 557, 514 Annotation: Subtype: Link Rect: 391, 480, 429, 491 Annotation: Subtype: Link Rect: 154, 458, 191, 468 Annotation: Subtype: Link Rect: 59, 435, 96, 445 Annotation: Subtype: Link Rect: 59, 400, 96, 410 Annotation: Subtype: Link Rect: 131, 365, 168, 376 Annotation: Subtype: Link Rect: 86, 342, 124, 353 Annotation: Subtype: Link Rect: 145, 319, 182, 330 Annotation: Subtype: Link Rect: 113, 285, 151, 295 Annotation: Subtype: Link Rect: 109, 262, 146, 272 Annotation: Subtype: Link Rect: 59, 227, 96, 238 Annotation: Subtype: Link Rect: 198, 204, 236, 215 Annotation: Subtype: Link Rect: 429, 181, 467, 192 Annotation: Subtype: Link Rect: 275, 157, 312, 168 Annotation: Subtype: Link Rect: 264, 123, 301, 134 Annotation: Subtype: Link Rect: 274, 100, 312, 111 Annotation: Subtype: Link Rect: 321, 77, 358, 88 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 131, 722, 168, 733 Annotation: Subtype: Link Rect: 322, 699, 359, 710 Annotation: Subtype: Link Rect: 457, 676, 495, 687 Annotation: Subtype: Link Rect: 431, 652, 468, 664 Annotation: Subtype: Link Rect: 272, 629, 310, 640 Annotation: Subtype: Link Rect: 59, 596, 96, 606 Annotation: Subtype: Link Rect: 250, 572, 288, 583 Annotation: Subtype: Link Rect: 157, 537, 194, 548 Annotation: Subtype: Link Rect: 200, 514, 237, 525 Annotation: Subtype: Link Rect: 290, 491, 327, 502 Annotation: Subtype: Link Rect: 394, 468, 432, 479 Annotation: Subtype: Link Rect: 459, 445, 497, 456 Annotation: Subtype: Link Rect: 503, 445, 539, 456 Annotation: Subtype: Link Rect: 324, 422, 362, 433 Annotation: Subtype: Link Rect: 368, 422, 404, 433 Annotation: Subtype: Link Rect: 519, 399, 557, 410 Annotation: Subtype: Link Rect: 59, 389, 95, 399 Annotation: Subtype: Link Rect: 235, 365, 272, 376 Annotation: Subtype: Link Rect: 135, 320, 173, 330 Annotation: Subtype: Link Rect: 369, 296, 407, 307 Annotation: Subtype: Link Rect: 499, 284, 536, 295 Annotation: Subtype: Link Rect: 394, 261, 431, 272 Annotation: Subtype: Link Rect: 461, 238, 498, 249 Annotation: Subtype: Link Rect: 393, 204, 431, 215 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 127, 159, 164, 169 Annotation: Subtype: Link Rect: 186, 135, 224, 146 Annotation: Subtype: Link Rect: 448, 112, 486, 123 Annotation: Subtype: Link Rect: 345, 89, 383, 100 Annotation: Subtype: Link Rect: 314, 66, 352, 77 Page: Sequence: 25 Annotations: Annotation: Subtype: Link Rect: 481, 722, 518, 733 Annotation: Subtype: Link Rect: 59, 699, 96, 710 Annotation: Subtype: Link Rect: 102, 699, 138, 710 Annotation: Subtype: Link Rect: 226, 664, 263, 675 Annotation: Subtype: Link Rect: 270, 664, 306, 675 Annotation: Subtype: Link Rect: 416, 641, 453, 652 Annotation: Subtype: Link Rect: 460, 641, 496, 652 Annotation: Subtype: Link Rect: 344, 618, 381, 629 Annotation: Subtype: Link Rect: 388, 618, 424, 629 Annotation: Subtype: Link Rect: 59, 584, 96, 594 Annotation: Subtype: Link Rect: 520, 560, 557, 572 Annotation: Subtype: Link Rect: 59, 550, 95, 560 Annotation: Subtype: Link Rect: 494, 526, 532, 537 Annotation: Subtype: Link Rect: 271, 503, 308, 514 Annotation: Subtype: Link Rect: 283, 469, 320, 479 Annotation: Subtype: Link Rect: 154, 445, 192, 456 Checksum: b5b47ecf Type: CRC32 Checksum: 76a1112b8641c12fffafae0f5535027e Type: MD5 Checksum: b4a5e175d21365bf6c75522758fb98f336501d09 Type: SHA-1 Checksum: 8185932c830c4b51c0a3bda09ae765fafd0085516fe5f766da118d3832a9ce97 Type: SHA-256