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Lactic acid is a sperm motility inactivation factor in the sperm storage tubules.

Matsuzaki M, Mizushima S, Hiyama G, Hirohashi N, Shiba K, Inaba K, Suzuki T, Dohra H, Ohnishi T, Sato Y, Kohsaka T, Ichikawa Y, Atsumi Y, Yoshimura T, Sasanami T - Sci Rep (2015)

Bottom Line: In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract.Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs.Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0).

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Shizuoka 422-8529, Japan.

ABSTRACT
Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41°C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (

No MeSH data available.


Related in: MedlinePlus

Purification of a bioactive substance from utero-vaginal junction extracts.(a) Utero-vaginal junction (UVJ) extracts were loaded onto a Superdex 200 pg column. The detectable peak was collected and each fraction was evaporated to dryness, dissolved in a physiological saline (pH 7.4), and applied to the sperm motility assay. The bioactive fraction is indicated by the horizontal bar. (b) HPLC profile of bioactive fractions (in panel a) on a C-22 reverse-phase column. Each collected fraction is indicated with a horizontal bar and a numeral. (c) Effects of HPLC fractions on sperm motility. Ejaculated sperm were suspended in a medium containing each fraction (1% (v/v)) and the effects of each fraction on sperm motility were evaluated. Bioactivity was detected in fractions 2 and 3. Asterisk indicates a significant difference from H2O. (d) Thin layer chromatography (TLC) analysis of the bioactive substance. A mixture of fractions 2 and 3 (in panel b) was further separated through preparative TLC. The motility score for sperm in each fraction are indicated below the TLC photograph. White arrow indicates the bioactive substance. S, mixture of fractions 2 and 3.
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f1: Purification of a bioactive substance from utero-vaginal junction extracts.(a) Utero-vaginal junction (UVJ) extracts were loaded onto a Superdex 200 pg column. The detectable peak was collected and each fraction was evaporated to dryness, dissolved in a physiological saline (pH 7.4), and applied to the sperm motility assay. The bioactive fraction is indicated by the horizontal bar. (b) HPLC profile of bioactive fractions (in panel a) on a C-22 reverse-phase column. Each collected fraction is indicated with a horizontal bar and a numeral. (c) Effects of HPLC fractions on sperm motility. Ejaculated sperm were suspended in a medium containing each fraction (1% (v/v)) and the effects of each fraction on sperm motility were evaluated. Bioactivity was detected in fractions 2 and 3. Asterisk indicates a significant difference from H2O. (d) Thin layer chromatography (TLC) analysis of the bioactive substance. A mixture of fractions 2 and 3 (in panel b) was further separated through preparative TLC. The motility score for sperm in each fraction are indicated below the TLC photograph. White arrow indicates the bioactive substance. S, mixture of fractions 2 and 3.

Mentions: Tissue from the utero-vaginal junction (UVJ), an area where SSTs are present, was homogenized and ultrafiltrated (>10 kDa cutoff) to obtain UVJ extracts. The flow-through fraction was found to strongly suppress sperm motility in vitro (Supplementary Movies 1 and 2), whereas the fraction retained on the ultrafiltration membrane (i.e. >10 kDa) had no significant inhibitory effect on sperm motility; thus, the flow-through fraction was pooled for subsequent procedures. The bioactive substance was further purified by liquid chromatography using size-exclusion (Fig. 1a) and C-22 reverse phase (Fig. 1b,c) columns followed by preparative thin-layer chromatography (TLC) (Fig. 1d). Analytical TLC of the sample in the final step of purification detected only one major spot (Fig. 1d, arrow), which was thereafter assigned as lactic acid by the spectrum analysis (see Methods). To determine the concentrations of L-lactic acid in SSTs, we used laser microdissection to isolate SST epithelial cells and non-SST epithelial cells (the ciliated epithelial cells that cover the surface of the UVJ) from frozen sections of UVJ. This method minimizes loss of small molecules. Although the epithelial lining of the SSTs or the surface epithelium was not clearly visible on the frozen sections without fixation and staining, the SSTs were easily distinguished by a unique ring- or tube-like structure (Fig. 2b). Extracts of the epithelial cells were assayed for L-lactic acid, which was found to be five times greater in SST epithelial cells (14 ± 3.4 mM, n = 4) than in non-SST epithelial cells (2.9 ± 0.6 mM, n = 4, Fig. 2a–c).


Lactic acid is a sperm motility inactivation factor in the sperm storage tubules.

Matsuzaki M, Mizushima S, Hiyama G, Hirohashi N, Shiba K, Inaba K, Suzuki T, Dohra H, Ohnishi T, Sato Y, Kohsaka T, Ichikawa Y, Atsumi Y, Yoshimura T, Sasanami T - Sci Rep (2015)

Purification of a bioactive substance from utero-vaginal junction extracts.(a) Utero-vaginal junction (UVJ) extracts were loaded onto a Superdex 200 pg column. The detectable peak was collected and each fraction was evaporated to dryness, dissolved in a physiological saline (pH 7.4), and applied to the sperm motility assay. The bioactive fraction is indicated by the horizontal bar. (b) HPLC profile of bioactive fractions (in panel a) on a C-22 reverse-phase column. Each collected fraction is indicated with a horizontal bar and a numeral. (c) Effects of HPLC fractions on sperm motility. Ejaculated sperm were suspended in a medium containing each fraction (1% (v/v)) and the effects of each fraction on sperm motility were evaluated. Bioactivity was detected in fractions 2 and 3. Asterisk indicates a significant difference from H2O. (d) Thin layer chromatography (TLC) analysis of the bioactive substance. A mixture of fractions 2 and 3 (in panel b) was further separated through preparative TLC. The motility score for sperm in each fraction are indicated below the TLC photograph. White arrow indicates the bioactive substance. S, mixture of fractions 2 and 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664960&req=5

f1: Purification of a bioactive substance from utero-vaginal junction extracts.(a) Utero-vaginal junction (UVJ) extracts were loaded onto a Superdex 200 pg column. The detectable peak was collected and each fraction was evaporated to dryness, dissolved in a physiological saline (pH 7.4), and applied to the sperm motility assay. The bioactive fraction is indicated by the horizontal bar. (b) HPLC profile of bioactive fractions (in panel a) on a C-22 reverse-phase column. Each collected fraction is indicated with a horizontal bar and a numeral. (c) Effects of HPLC fractions on sperm motility. Ejaculated sperm were suspended in a medium containing each fraction (1% (v/v)) and the effects of each fraction on sperm motility were evaluated. Bioactivity was detected in fractions 2 and 3. Asterisk indicates a significant difference from H2O. (d) Thin layer chromatography (TLC) analysis of the bioactive substance. A mixture of fractions 2 and 3 (in panel b) was further separated through preparative TLC. The motility score for sperm in each fraction are indicated below the TLC photograph. White arrow indicates the bioactive substance. S, mixture of fractions 2 and 3.
Mentions: Tissue from the utero-vaginal junction (UVJ), an area where SSTs are present, was homogenized and ultrafiltrated (>10 kDa cutoff) to obtain UVJ extracts. The flow-through fraction was found to strongly suppress sperm motility in vitro (Supplementary Movies 1 and 2), whereas the fraction retained on the ultrafiltration membrane (i.e. >10 kDa) had no significant inhibitory effect on sperm motility; thus, the flow-through fraction was pooled for subsequent procedures. The bioactive substance was further purified by liquid chromatography using size-exclusion (Fig. 1a) and C-22 reverse phase (Fig. 1b,c) columns followed by preparative thin-layer chromatography (TLC) (Fig. 1d). Analytical TLC of the sample in the final step of purification detected only one major spot (Fig. 1d, arrow), which was thereafter assigned as lactic acid by the spectrum analysis (see Methods). To determine the concentrations of L-lactic acid in SSTs, we used laser microdissection to isolate SST epithelial cells and non-SST epithelial cells (the ciliated epithelial cells that cover the surface of the UVJ) from frozen sections of UVJ. This method minimizes loss of small molecules. Although the epithelial lining of the SSTs or the surface epithelium was not clearly visible on the frozen sections without fixation and staining, the SSTs were easily distinguished by a unique ring- or tube-like structure (Fig. 2b). Extracts of the epithelial cells were assayed for L-lactic acid, which was found to be five times greater in SST epithelial cells (14 ± 3.4 mM, n = 4) than in non-SST epithelial cells (2.9 ± 0.6 mM, n = 4, Fig. 2a–c).

Bottom Line: In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract.Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs.Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0).

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Shizuoka 422-8529, Japan.

ABSTRACT
Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41°C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (

No MeSH data available.


Related in: MedlinePlus