capacitation hyperactivation acrosome hyperactivation AR bovine serum albumin BSA non-genomic effect isothiocyanate; FITC PR mrna P hyperactivation HA

Transcription

1 P PRP total RNA cdna PCR primer set PR mrna P hyperactivation HA AR Ca PR P HA AR P Ca PR mrna P DNA C PR PR P P HA AR Ca mrna capacitation hyperactivation acrosome reaction; AR hyperactivation AR Ca Ca ,2,6,7 Ca 1 bovine serum albumin BSA 8 non-genomic effect fluorescein isothiocyanate; FITC 9 Ca

2 48 PR PR PR hyperactivation AR Ca 1 HTF Sigma Chemical Co., Cat. No. 9962, Poole Dorset, UK Chirgwin et al. 10 total RNA 4 M 5.7 M Reverse transcription-polymerase chain reaction RT-PCR total RNA 200 ng random primer Takara, Kyoto, Japan RAV-2 reverse transcriptase cdna cdna template mm MgCl Taq DNA polymerase 94 C 1 55 C 1 72 C P15 forward P35 reverse 5 -AGGAGTTTGT- CAAGCTTCAA-3 5 -GGGACTGGATAAATG- TATTC-3 PR cdna 11 exon A B C mrna P36 forward P33 reverse 5 - CCCTCCGACGAAAAGACGC-3 5 -AAC- 1 isoform A, B, C

3 49 CTTGCACCCGGACCGGCTCA-3 DNA A/B A B mrna Southern blotting 5 µl P-labeled PR cdna probe WHO C 5 CO human serum albumin HSA; Sigma Chemical Co., St. Louis, MO, U.S.A. modified Biggers, Whitten and Whittinghams mbww 13 swim up C 5 CO 2 1 nm 10 nm 100 nm 1 µm mbww mbww 30 hyperactivated sperm Sigma Chemical Co. dimethylsulfoxide DMSO; Sigma Chemical Co. DMSO 1 mbww Hyperactivation Hamilton- Thorne Research Motility Analyzer HTM-IVOS, version 10, Beverly, MA, U.S.A. track velocity 100 microns/sec linearity 65 maximum amplitude for lateral displacement 7.5 microns hyperactivated sperm Burkman swim up mbww 6 10 nm 100 nm 500 nm 1 µm 3 µm 15 µm 30 µm mbww 30 acrosomereacted sperm Cross 16,17 Hoechst Sigma Chemical Co. 10 µg/ml fluorescein isothiocyanate-conjugated pisum sativum agglutinin FITC-PSA; Sigma Chemical Co. 30 Olympus BHT; Olympus, Tokyo Hoechst ARAcrosome cap FITC-PSA acrosome intact sperm equatrial segment acrosome-reacted sperm g 1 postestrous discharge pregnant mare s serum PMS; Sigma Chemical Co. 30 IU 3 human chorionic gonadotropin HCG; Mochida, Tokyo 30 IU HCG hyaluronidase Sigma Chemical Co. 15 mbww trypsin Sigma Chemical Co. mbww 3 mbww swim up

4 50 mbww 6 10 nm 100 nm 500 nm 1 µm 3 µm 15 µm 30 µm 1 ml mbww 30 mbww HSA mbww 300 µl Petri dish /ml 37 C 5 CO 2 4 Olympus BX, Olympus, Tokyo 5 Ca free mbww AR Ca 15 µm 5 Hyperactivation AR t χ2 1 A B C PR mrna exon P15/35 DNA A-B P36/ nm 2B A C mrna B A mrna A PR B A C mrna B PR B A mrna C β actin mrna Lane 1, ejaculated spermatozoa. Lane 2, proliferative phase uterine endometrium 200 ng. Lane 3, 50 ng. Lane 4, 13 ng. Lane 5, 3.1 ng. Lane 6, 0.78 ng. Lane 7, 0.20 ng. Lane 8, 0.05 ng. Lane 9, peripheral blood leucocyte. Lane 10, RT-PCR blank.

5 51 hyperactivated sperm p nm 3 µm acrosome-reacted sperm 15 µm 30 µm acrosome-reacted sperm p nm mm Ca 15 µm AR 3 Ca 15 µm acrosome-re- Progesterone 1 hyperactivation Progesterone n 8 Hyperactivated sperm mean SD 0nM nM nM nM * 1000nM ** **p 0.01 vs. 0nM 2 AR Acrosome-reacted sperm mean SD *p 0.05 vs. 0nM Hamster test of eggs penetrated 0nM nM nM nM * 1 µm ** 3 µm ** 15 µm ** 55.0** 30 µm ** 86.4** **p 0.01 vs. 0 µm 3 AR Ca *p 0.05 vs. 0 µm Ca 2+ Progesterone Acrosome-reacted sperm Hamster test mean SD of eggs penetrated 0mM 0 µm mM 15 µm * * 10.2 ** * * ** ** ** 1.71mM 0 µm mM 15 µm * 53.4 ** **p 0.01 *p 0.05

6 52 acted sperm p 0.05 p 0.01Ca-free Ca acrosome-reacted sperm p 0.05 p 0.01 hyperactivation AR 5 Ca 1,2,6,7 protein kinase C 19 PR PR 20 PR A B C 3 8 exon PR B mrna A mrna exon 1 C mrna exon 2 B A C PR mrna B A PR mrna RT-PCR mrna PR mrna mrna B A PR mrna DNA C PR PR mrna Uhler hyperactivation 10 nm hyperactivated sperm nm hyperactivated sperm hyperactivation Meizel AR 250 ng/ml 800 nm AR 22 Brucker 6 15 µm AR 2-3 µm Meizel AR AR µm

7 53 hyperactivation capacitation AR AR AR Hyperactivation AR Ca 1,2,6,7 Ca AR 25 Ca AR 26 Ca Ca Ca Ca channel blocker AR 27 Ca Ca PR Ca Ca Ca capacitation hyperactivation Ca ZP 3 Ca P Ca 1) Yanagimachi R: Mammalian fertilization. In: Knobil E, Neill JD, Eds. Physiology of Reproduction. New York; Ranvan Press, p , ) Blackmore PF et al: Progesterone and 17 alphahydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem, 265: , ) Thomas P and Meizel S: An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gemete Res, 20: , ) Fabbri R et al: Follicular fluid and human granulosa cell cultures: influence on sperm kinetic parameters, hyperactivation, and acrosome reaction. Fertil Steril, 69: , ) Uhler ML: Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril, 58: , ) Brucker C et al: Progesterone-induced acrosome reaction: potential role for sperm acrosome reaction antigen-1 in fertilization. Hum Reprod, 9: , ) Fukui K et al: The effect of follicular fluid on intracellular free calcium levels in human spermatozoa. Asia-Oceania J Obstet Gynecol, 21: 43 49, ) Meizel S and Turner KO: Progesterone acts at the plasma membrane of human sperm. Mol Cell Endocrinol, 77: R1 R5, ) Tesarik J et al: Selective expression of a progesterone receptor on the human sperm surface. Fertil Steril, 58: , ) Chirgwin JM et al: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem, 18: , ) Kastner P et al: Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J, 9: , ) World Health Organization: WHO laboratory manual for the examination of human semen and semen-cervical mucus interactions. Cambridge, 3 26, The Press Syndicate of Cambridge Univ, ) Biggers JD et al: Methods of mammalian embryology, Eds. Daniel JD, p , New York; Freeman & Co, ) Sperm preparation AIH 1: 39 45, ) Burkman LJ: Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil Steril, 55: , ) Cross NL et al: Two simple methods for detecting acrosome-reacted human sperm. Gamete Res,

8 54 15: , ) Mendoza C et al: Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil, 95: , ) Yanagimachi R: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod, 15: , ) Bonaccorsi L et al: Progesterone-stimulated intracellular calcium increase in human spermatozoa is protein kinase C-independent. Mol Hum Reprod, 4: , ) Mahesh VB et al: Diverse modes of action of progesterone and its metabolites. J Steroid Biochem Mol Biol, 56: , ) Uhler ML et al: Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril, 58: , ) Meizel S et al: Fertilization in Mammals, Eds. Bavister BD et al, p , Serono Symposia USA, Norwell, Massachusetts, ) Osman RA et al: Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commum, 160: , ) Cross NL et al: Induction of acrosome reaction by the human zona pellucida. Biol Reprod, 38: , ) Didion BA and Graves CN: Influence of calcium on the true acrosome reaction and capacity of bull spermatozoa to penetrate oocytes in vitro. J Dairy Sci, 72: , ) Effects of calcium and magnesium on in vitro fertilization in human 34: , ) Goodwin LO et al: Isolation and characterization of the primary structure of testis-specific L-type calcium channel: implications for contraception. Mol Hum Reprod, 3: , 1997.