Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels
The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing age...
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Published in | Biology of reproduction Vol. 31; no. 4; pp. 797 - 809 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Madison, WI
Society for the Study of Reproduction
01.11.1984
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Subjects | |
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Abstract | The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using
both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups.
In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide
(NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed
on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0.
Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation;
thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence
microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining
both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa
reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free
modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium.
However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in
the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed
by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment
caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates
of the SS/SH ratio. |
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AbstractList | The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using
both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups.
In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide
(NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed
on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0.
Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation;
thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence
microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining
both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa
reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free
modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium.
However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in
the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed
by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment
caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates
of the SS/SH ratio. The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide (NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0. Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation; thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium. However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates of the SS/SH ratio. |
Author | T T Huang N S Kosower R Yanagimachi |
Author_xml | – sequence: 1 givenname: T. T. F surname: HUANG fullname: HUANG, T. T. F organization: Univ. Hawai, school medicine, Honolulu HI 96822, United States – sequence: 2 givenname: N. S surname: KOSOWER fullname: KOSOWER, N. S organization: Univ. Hawai, school medicine, Honolulu HI 96822, United States – sequence: 3 givenname: R surname: YANAGIMACHI fullname: YANAGIMACHI, R organization: Univ. Hawai, school medicine, Honolulu HI 96822, United States |
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Keywords | Ripening Vertebrata Spermatozoid capacitation Organic disulfide Spermatozoa Thiol Mammalia Guinea pig Rodentia Fluorescence Tagging Localization |
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Snippet | The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using
both membrane-permeable (mBBr)... The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr)... |
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SubjectTerms | Animals Biological and medical sciences Bridged Bicyclo Compounds Disulfides - metabolism Fluorescent Dyes Fundamental and applied biological sciences. Psychology Guinea Pigs Histocytochemistry Male Mammalian male genital system Morphology. Physiology Quaternary Ammonium Compounds Sperm Capacitation Sperm Maturation Spermatozoa - metabolism Sulfhydryl Compounds - metabolism Vertebrates: reproduction |
Title | Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels |
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