PMID- 22552691
OWN - NLM
STAT- MEDLINE
DCOM- 20121113
LR  - 20141120
IS  - 1460-2350 (Electronic)
IS  - 0268-1161 (Linking)
VI  - 27
IP  - 7
DP  - 2012 Jul
TI  - Heparin enhances protamine disulfide bond reduction during in vitro 
      decondensation of human spermatozoa.
PG  - 1930-8
LID - 10.1093/humrep/des139 [doi]
AB  - BACKGROUND: Human sperm nuclear decondensation in vivo involves protamine 
      disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, 
      presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the 
      present study was to test the hypothesis that these two events occur 
      simultaneously rather than sequentially, as has been hitherto accepted, and to 
      test for the presence of HS in the human oocyte. METHODS: Spermatozoa and 
      isolated sperm nuclei obtained from normal volunteers were exposed in vitro to 
      heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as 
      the disulfide reducing agent. Decondensing reagents were added either 
      simultaneously or sequentially. Percentage sperm nuclear decondensation was 
      assayed by phase contrast microscopy. Thiol reduced status of isolated sperm 
      nuclei was evaluated both indirectly [acridine orange (AO) staining of 
      acid-denatured DNA] and directly [monobromobimane (mBBr) staining of 
      protamine-free thiols]. The presence of HS in mature metaphase II (MII) human 
      oocytes was analyzed by immunocytochemistry. RESULTS: Sequential addition of 
      reagents always resulted in significantly lower decondensation if GSH was used as 
      the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but 
      only when heparin was used first, when DTT was the disulfide reducing agent (P < 
      0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr 
      staining of protamines revealed that the addition of heparin to GSH but not to 
      DTT significantly increased the thiol reduced status of sperm chromatin. HS was 
      detected in the ooplasm of zona-free MII human oocytes. CONCLUSIONS: The results 
      presented in this paper clearly show that heparin enhances the sperm chromatin 
      thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction 
      and protamine/histone exchange could occur as simultaneous, rather than 
      sequential, events. We also demonstrate for the first time the presence of HS in 
      the human oocyte.
FAU - Julianelli, V
AU  - Julianelli V
AD  - Instituto de Biologia y Medicina Experimental (IBYME)-CONICET, Vuelta de Obligado 
      2490, Buenos Aires C1428ADN, Argentina.
FAU - Farrando, B
AU  - Farrando B
FAU - Alvarez Sedo, C
AU  - Alvarez Sedo C
FAU - Calvo, L
AU  - Calvo L
FAU - Romanato, M
AU  - Romanato M
FAU - Calvo, J C
AU  - Calvo JC
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120502
PL  - England
TA  - Hum Reprod
JT  - Human reproduction (Oxford, England)
JID - 8701199
RN  - 0 (Disulfides)
RN  - 0 (Protamines)
RN  - 0 (Sulfhydryl Compounds)
RN  - 9005-49-6 (Heparin)
RN  - GAN16C9B8O (Glutathione)
RN  - T8ID5YZU6Y (Dithiothreitol)
SB  - IM
MH  - Cell Nucleus/metabolism
MH  - Disulfides
MH  - Dithiothreitol/pharmacology
MH  - Female
MH  - Glutathione/metabolism
MH  - Heparin/chemistry/*pharmacology
MH  - Humans
MH  - Immunohistochemistry/methods
MH  - In Vitro Techniques
MH  - Male
MH  - Microscopy, Phase-Contrast/methods
MH  - Oocytes/cytology
MH  - Protamines/*chemistry
MH  - Spermatozoa/*metabolism
MH  - Sulfhydryl Compounds/chemistry
MH  - Time Factors
EDAT- 2012/05/04 06:00
MHDA- 2012/11/14 06:00
CRDT- 2012/05/04 06:00
PHST- 2012/05/04 06:00 [entrez]
PHST- 2012/05/04 06:00 [pubmed]
PHST- 2012/11/14 06:00 [medline]
AID - des139 [pii]
AID - 10.1093/humrep/des139 [doi]
PST - ppublish
SO  - Hum Reprod. 2012 Jul;27(7):1930-8. doi: 10.1093/humrep/des139. Epub 2012 May 2.