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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

Sperm storage tubules produced large quantities of lactic acid.(a) Lactic acid levels in sperm storage tubule (SST) epithelium extracts and surface epithelial cells (Non-SST epithelium) isolated by laser microdissection (LMD). Values shown are means ± SEM of four independent experiments. Lactic acid levels were significantly greater in the SST epithelium than in the Non-SST epithelium (P < 0.05). (b,c) Photographs of a cryosection of the utero-vaginal junction (UVJ) before (b) and after (c) LMD. Arrows and arrowheads in panel b indicate SSTs and Non-SST epithelium, respectively. Scale bar = 150 μm. (d) Photograph of SSTs isolated by collagenase digestion. Scale bar = 100 μm. (e) Photograph of non-SST cells isolated by collagenase digestion. Scale bar = 200 μm. Arrows and the arrowhead indicate surface epithelial cells and lamina propria, respectively. (f) Time-dependent release of lactic acid by cultured SSTs. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc) or 1 mM 2-deoxyglucose (2-DOG). The medium was sampled at the indicated times and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Asterisks indicate significant differences from Glc at each incubation time (P < 0.05). (g) SSTs produced lactic acid via glycolysis. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc), 1 mM 2-deoxyglucose (2-DOG) or 1 mM 2-deoxygalactose (2-DOGal). The medium was sampled at 60 min of incubation and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.01). (h) Expression of monocarboxylate transporter mRNA. mRNA extracted from the UVJ was reverse transcribed, and an aliquot was subjected to PCR using the primer set (MCT1, MCT2, MTC3, MTC4 or S17) indicated at the top of the figure. For a non-RT control, mRNA from the UVJ was treated the same way, except reverse transcriptases (lanes -) were omitted. The representative gel of three independent experiments was shown. M, molecular weight marker.
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f2: Sperm storage tubules produced large quantities of lactic acid.(a) Lactic acid levels in sperm storage tubule (SST) epithelium extracts and surface epithelial cells (Non-SST epithelium) isolated by laser microdissection (LMD). Values shown are means ± SEM of four independent experiments. Lactic acid levels were significantly greater in the SST epithelium than in the Non-SST epithelium (P < 0.05). (b,c) Photographs of a cryosection of the utero-vaginal junction (UVJ) before (b) and after (c) LMD. Arrows and arrowheads in panel b indicate SSTs and Non-SST epithelium, respectively. Scale bar = 150 μm. (d) Photograph of SSTs isolated by collagenase digestion. Scale bar = 100 μm. (e) Photograph of non-SST cells isolated by collagenase digestion. Scale bar = 200 μm. Arrows and the arrowhead indicate surface epithelial cells and lamina propria, respectively. (f) Time-dependent release of lactic acid by cultured SSTs. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc) or 1 mM 2-deoxyglucose (2-DOG). The medium was sampled at the indicated times and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Asterisks indicate significant differences from Glc at each incubation time (P < 0.05). (g) SSTs produced lactic acid via glycolysis. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc), 1 mM 2-deoxyglucose (2-DOG) or 1 mM 2-deoxygalactose (2-DOGal). The medium was sampled at 60 min of incubation and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.01). (h) Expression of monocarboxylate transporter mRNA. mRNA extracted from the UVJ was reverse transcribed, and an aliquot was subjected to PCR using the primer set (MCT1, MCT2, MTC3, MTC4 or S17) indicated at the top of the figure. For a non-RT control, mRNA from the UVJ was treated the same way, except reverse transcriptases (lanes -) were omitted. The representative gel of three independent experiments was shown. M, molecular weight marker.

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)

Sperm storage tubules produced large quantities of lactic acid.(a) Lactic acid levels in sperm storage tubule (SST) epithelium extracts and surface epithelial cells (Non-SST epithelium) isolated by laser microdissection (LMD). Values shown are means ± SEM of four independent experiments. Lactic acid levels were significantly greater in the SST epithelium than in the Non-SST epithelium (P < 0.05). (b,c) Photographs of a cryosection of the utero-vaginal junction (UVJ) before (b) and after (c) LMD. Arrows and arrowheads in panel b indicate SSTs and Non-SST epithelium, respectively. Scale bar = 150 μm. (d) Photograph of SSTs isolated by collagenase digestion. Scale bar = 100 μm. (e) Photograph of non-SST cells isolated by collagenase digestion. Scale bar = 200 μm. Arrows and the arrowhead indicate surface epithelial cells and lamina propria, respectively. (f) Time-dependent release of lactic acid by cultured SSTs. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc) or 1 mM 2-deoxyglucose (2-DOG). The medium was sampled at the indicated times and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Asterisks indicate significant differences from Glc at each incubation time (P < 0.05). (g) SSTs produced lactic acid via glycolysis. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc), 1 mM 2-deoxyglucose (2-DOG) or 1 mM 2-deoxygalactose (2-DOGal). The medium was sampled at 60 min of incubation and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.01). (h) Expression of monocarboxylate transporter mRNA. mRNA extracted from the UVJ was reverse transcribed, and an aliquot was subjected to PCR using the primer set (MCT1, MCT2, MTC3, MTC4 or S17) indicated at the top of the figure. For a non-RT control, mRNA from the UVJ was treated the same way, except reverse transcriptases (lanes -) were omitted. The representative gel of three independent experiments was shown. M, molecular weight marker.
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f2: Sperm storage tubules produced large quantities of lactic acid.(a) Lactic acid levels in sperm storage tubule (SST) epithelium extracts and surface epithelial cells (Non-SST epithelium) isolated by laser microdissection (LMD). Values shown are means ± SEM of four independent experiments. Lactic acid levels were significantly greater in the SST epithelium than in the Non-SST epithelium (P < 0.05). (b,c) Photographs of a cryosection of the utero-vaginal junction (UVJ) before (b) and after (c) LMD. Arrows and arrowheads in panel b indicate SSTs and Non-SST epithelium, respectively. Scale bar = 150 μm. (d) Photograph of SSTs isolated by collagenase digestion. Scale bar = 100 μm. (e) Photograph of non-SST cells isolated by collagenase digestion. Scale bar = 200 μm. Arrows and the arrowhead indicate surface epithelial cells and lamina propria, respectively. (f) Time-dependent release of lactic acid by cultured SSTs. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc) or 1 mM 2-deoxyglucose (2-DOG). The medium was sampled at the indicated times and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Asterisks indicate significant differences from Glc at each incubation time (P < 0.05). (g) SSTs produced lactic acid via glycolysis. Isolated SSTs were cultured in Hank’s balanced salt solution containing either 5 mM glucose (Glc), 1 mM 2-deoxyglucose (2-DOG) or 1 mM 2-deoxygalactose (2-DOGal). The medium was sampled at 60 min of incubation and the lactic acid levels of the samples were measured. Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.01). (h) Expression of monocarboxylate transporter mRNA. mRNA extracted from the UVJ was reverse transcribed, and an aliquot was subjected to PCR using the primer set (MCT1, MCT2, MTC3, MTC4 or S17) indicated at the top of the figure. For a non-RT control, mRNA from the UVJ was treated the same way, except reverse transcriptases (lanes -) were omitted. The representative gel of three independent experiments was shown. M, molecular weight marker.
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