title: Fertilization defects in sperm from Cysteine-rich secretory protein 2 (Crisp2) knockout mice: implications for fertility disorders.
abstract: OBJECTIVE
  We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization.
  
  
  RESULTS
  Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation.
  
  
  BACKGROUND
  Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility.
  
  
  METHODS
  A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry).
  
  
  RESULTS
  Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001).
  
  
  CONCLUSIONS
  The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies.
  
  
  CONCLUSIONS
  Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility.
  
  
  BACKGROUND
  This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest.
authors.affiliation: Instituto de Biología y Medicina Experimental (IByME-CONICET), Ciudad Autónoma de Buenos Aires C1428ADN, Argentina.
authors.forename: N G
authors.initials: NG
authors.lastname: Brukman
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Miyata
authors.affiliation: Instituto de Investigación y Tecnología en Reproducción Animal, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires C1427CWO, Argentina.
authors.forename: P
authors.initials: P
authors.lastname: Torres
authors.affiliation: Instituto de Investigación y Tecnología en Reproducción Animal, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires C1427CWO, Argentina.
authors.forename: D
authors.initials: D
authors.lastname: Lombardo
authors.affiliation: Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), Departamento de Química Biológica (FCEN-UBA), Ciudad Autónoma de Buenos Aires C1405BWE, Argentina.
authors.forename: J J
authors.initials: JJ
authors.lastname: Caramelo
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ikawa
authors.affiliation: Instituto de Biología y Medicina Experimental (IByME-CONICET), Ciudad Autónoma de Buenos Aires C1428ADN, Argentina.
authors.forename: V G
authors.initials: VG
authors.lastname: Da Ros
authors.affiliation: Instituto de Biología y Medicina Experimental (IByME-CONICET), Ciudad Autónoma de Buenos Aires C1428ADN, Argentina pcuasnicu@ibyme.conicet.gov.ar.
authors.forename: P S
authors.initials: PS
authors.lastname: Cuasnicú
mesh_terms: D000328:Adult; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules; D004971:Estrus / Q000235:genetics; D005091:Exons; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000172:deficiency / Q000235:genetics*; D006801:Humans; D007248:Infertility, Male / Q000235:genetics* / Q000503:physiopathology / Q000601:surgery; D008098:Litter Size; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D017384:Sequence Deletion; D013075:Sperm Capacitation / Q000235:genetics; D013084:Sperm-Ovum Interactions / Q000235:genetics*; D013094:Spermatozoa / Q000378:metabolism* / Q000473:pathology; D014659:Vasectomy; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: C062621:CRISP2 protein, human; D015815:Cell Adhesion Molecules; C483816:Crisp2 protein, mouse; D006023:Glycoproteins; D008565:Membrane Proteins; D002118:Calcium
keywords: CRISP; fertility; fertilization; hyperactivation; knockout mice; motility; sperm; testes