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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

Mechanism of sperm motility inactivation that resulted from exposure to lactic acid.(a) Effects of lactic acid on sperm motility in vitro. Ejaculated sperm were suspended in media containing various concentrations of either L-lactic acid or D-lactic acid, and sperm motility was evaluated on a scale of 0 to 5. Values shown are means ± SEM of three independent experiments. *P < 0.05 vs. 0 mM. **P < 0.01 vs. 0 mM. (b) Changes in the pHe or pHi of sperm in response to the addition of L-lactic acid. pHe of the medium was directly measured using a pH meter. Sperm pHi was determined using pHrodoTM. The results shown are representative of results from five independent experiments. (c) ATP levels in sperm incubated with L-lactic acid. ATP concentrations of sperm are expressed as mean values ± SEM of three independent experiments. (d,e) Photographs of pHrodoTM-loaded sperm swimming in Hank’s balanced salt solution (d) and resident sperm in SSTs (e). Dashed line indicates the outline of SSTs. Scale bar = 50 μm. (f) Representative standard curve of sperm pHi equilibrated with the indicated pH buffer containing valinomycin and nigericin. (g) pHi values of sperm swimming in Hank’s balanced salt solution (Extender) and resident sperm in SSTs (SSTs). Values shown are means ± SEM of three independent experiments. Asterisk denotes a significant difference (P < 0.00001). (h) Effects of pH on ATPase activity of sperm. De-membraned sperm were incubated with 1 mM ATP, and the free phosphoric acid in the incubation mixture was measured using a microplate reader. (i) Effects of pH on axonemal sliding of sperm. Demembraned sperm were mounted on chamber slides, treated with trypsin, and an assay buffer that adjusted the pH to either 7.4, 6.4, 5.7, or 5.4 was perfused for equilibration. The ATP solution was perfused and axonemal sliding was recorded. Percentages of axonemal sliding were calculated and expressed as mean values ± SEM of three independent experiments. **denotes a significant difference from pH 7.4 (P < 0.0000001).
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f4: Mechanism of sperm motility inactivation that resulted from exposure to lactic acid.(a) Effects of lactic acid on sperm motility in vitro. Ejaculated sperm were suspended in media containing various concentrations of either L-lactic acid or D-lactic acid, and sperm motility was evaluated on a scale of 0 to 5. Values shown are means ± SEM of three independent experiments. *P < 0.05 vs. 0 mM. **P < 0.01 vs. 0 mM. (b) Changes in the pHe or pHi of sperm in response to the addition of L-lactic acid. pHe of the medium was directly measured using a pH meter. Sperm pHi was determined using pHrodoTM. The results shown are representative of results from five independent experiments. (c) ATP levels in sperm incubated with L-lactic acid. ATP concentrations of sperm are expressed as mean values ± SEM of three independent experiments. (d,e) Photographs of pHrodoTM-loaded sperm swimming in Hank’s balanced salt solution (d) and resident sperm in SSTs (e). Dashed line indicates the outline of SSTs. Scale bar = 50 μm. (f) Representative standard curve of sperm pHi equilibrated with the indicated pH buffer containing valinomycin and nigericin. (g) pHi values of sperm swimming in Hank’s balanced salt solution (Extender) and resident sperm in SSTs (SSTs). Values shown are means ± SEM of three independent experiments. Asterisk denotes a significant difference (P < 0.00001). (h) Effects of pH on ATPase activity of sperm. De-membraned sperm were incubated with 1 mM ATP, and the free phosphoric acid in the incubation mixture was measured using a microplate reader. (i) Effects of pH on axonemal sliding of sperm. Demembraned sperm were mounted on chamber slides, treated with trypsin, and an assay buffer that adjusted the pH to either 7.4, 6.4, 5.7, or 5.4 was perfused for equilibration. The ATP solution was perfused and axonemal sliding was recorded. Percentages of axonemal sliding were calculated and expressed as mean values ± SEM of three independent experiments. **denotes a significant difference from pH 7.4 (P < 0.0000001).

Mentions: The effects of exogenous lactic acid on sperm motility were then investigated in vitro. Sperm motility decreased in response to exposure to L-lactic acid in a dose-dependent manner, while D-lactic acid had a lesser inhibitory effect (Fig. 4a and Supplementary Movie 3), suggesting the involvement to some extent of molecules with stereospecificity such as lactate/pyruvate dehydrogenases. However, the sperm quiescence activity was not due to lactic acid itself because the addition of other organic acids such as acetic acid, malic acid, oxaloacetic acid, citric acid and even hydrochloric acid (pH 5) inactivated sperm motility (Supplementary Fig. S2 online). Notably, a strong correlation between extracellular pH and sperm quiescence was observed (R2 = 0.95, Supplementary Fig. S2 online), suggesting that the sperm immobilization is caused by lowering extracellular pH (pHe). The data showed that 10 mM lactic acid was sufficient to immobilize sperm, which is in line with the physiological concentrations found in SST extracts (i.e., 14 ± 3.4 mM). Because pHe decreased in response to the addition of lactic acid (Fig. 4b), we next examined whether lactic acid addition can decrease sperm intracellular pH (pHi). When pHrodo-loaded sperm were exposed to different concentrations of L-lactic acid, sperm pHi and pHe were concurrently decreased (Fig. 4b). On the other hand, intracellular ATP levels were not affected by the addition of L-lactic acid (Fig. 4c), which indicated that the lactic acid-induced sperm quiescence is not due to the depletion of intracellular ATP. Furthermore, a direct pHi measurement of SST-stored sperm, which was enabled by intra-vaginal insemination of pHrodo-loaded sperm, clearly demonstrated that intracellular acidosis of the resident sperm occurred (Fig. 4d,e,g, pH 6.2 ± 0.1, means ± SE, n = 29).


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)

Mechanism of sperm motility inactivation that resulted from exposure to lactic acid.(a) Effects of lactic acid on sperm motility in vitro. Ejaculated sperm were suspended in media containing various concentrations of either L-lactic acid or D-lactic acid, and sperm motility was evaluated on a scale of 0 to 5. Values shown are means ± SEM of three independent experiments. *P < 0.05 vs. 0 mM. **P < 0.01 vs. 0 mM. (b) Changes in the pHe or pHi of sperm in response to the addition of L-lactic acid. pHe of the medium was directly measured using a pH meter. Sperm pHi was determined using pHrodoTM. The results shown are representative of results from five independent experiments. (c) ATP levels in sperm incubated with L-lactic acid. ATP concentrations of sperm are expressed as mean values ± SEM of three independent experiments. (d,e) Photographs of pHrodoTM-loaded sperm swimming in Hank’s balanced salt solution (d) and resident sperm in SSTs (e). Dashed line indicates the outline of SSTs. Scale bar = 50 μm. (f) Representative standard curve of sperm pHi equilibrated with the indicated pH buffer containing valinomycin and nigericin. (g) pHi values of sperm swimming in Hank’s balanced salt solution (Extender) and resident sperm in SSTs (SSTs). Values shown are means ± SEM of three independent experiments. Asterisk denotes a significant difference (P < 0.00001). (h) Effects of pH on ATPase activity of sperm. De-membraned sperm were incubated with 1 mM ATP, and the free phosphoric acid in the incubation mixture was measured using a microplate reader. (i) Effects of pH on axonemal sliding of sperm. Demembraned sperm were mounted on chamber slides, treated with trypsin, and an assay buffer that adjusted the pH to either 7.4, 6.4, 5.7, or 5.4 was perfused for equilibration. The ATP solution was perfused and axonemal sliding was recorded. Percentages of axonemal sliding were calculated and expressed as mean values ± SEM of three independent experiments. **denotes a significant difference from pH 7.4 (P < 0.0000001).
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Related In: Results  -  Collection

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

f4: Mechanism of sperm motility inactivation that resulted from exposure to lactic acid.(a) Effects of lactic acid on sperm motility in vitro. Ejaculated sperm were suspended in media containing various concentrations of either L-lactic acid or D-lactic acid, and sperm motility was evaluated on a scale of 0 to 5. Values shown are means ± SEM of three independent experiments. *P < 0.05 vs. 0 mM. **P < 0.01 vs. 0 mM. (b) Changes in the pHe or pHi of sperm in response to the addition of L-lactic acid. pHe of the medium was directly measured using a pH meter. Sperm pHi was determined using pHrodoTM. The results shown are representative of results from five independent experiments. (c) ATP levels in sperm incubated with L-lactic acid. ATP concentrations of sperm are expressed as mean values ± SEM of three independent experiments. (d,e) Photographs of pHrodoTM-loaded sperm swimming in Hank’s balanced salt solution (d) and resident sperm in SSTs (e). Dashed line indicates the outline of SSTs. Scale bar = 50 μm. (f) Representative standard curve of sperm pHi equilibrated with the indicated pH buffer containing valinomycin and nigericin. (g) pHi values of sperm swimming in Hank’s balanced salt solution (Extender) and resident sperm in SSTs (SSTs). Values shown are means ± SEM of three independent experiments. Asterisk denotes a significant difference (P < 0.00001). (h) Effects of pH on ATPase activity of sperm. De-membraned sperm were incubated with 1 mM ATP, and the free phosphoric acid in the incubation mixture was measured using a microplate reader. (i) Effects of pH on axonemal sliding of sperm. Demembraned sperm were mounted on chamber slides, treated with trypsin, and an assay buffer that adjusted the pH to either 7.4, 6.4, 5.7, or 5.4 was perfused for equilibration. The ATP solution was perfused and axonemal sliding was recorded. Percentages of axonemal sliding were calculated and expressed as mean values ± SEM of three independent experiments. **denotes a significant difference from pH 7.4 (P < 0.0000001).
Mentions: The effects of exogenous lactic acid on sperm motility were then investigated in vitro. Sperm motility decreased in response to exposure to L-lactic acid in a dose-dependent manner, while D-lactic acid had a lesser inhibitory effect (Fig. 4a and Supplementary Movie 3), suggesting the involvement to some extent of molecules with stereospecificity such as lactate/pyruvate dehydrogenases. However, the sperm quiescence activity was not due to lactic acid itself because the addition of other organic acids such as acetic acid, malic acid, oxaloacetic acid, citric acid and even hydrochloric acid (pH 5) inactivated sperm motility (Supplementary Fig. S2 online). Notably, a strong correlation between extracellular pH and sperm quiescence was observed (R2 = 0.95, Supplementary Fig. S2 online), suggesting that the sperm immobilization is caused by lowering extracellular pH (pHe). The data showed that 10 mM lactic acid was sufficient to immobilize sperm, which is in line with the physiological concentrations found in SST extracts (i.e., 14 ± 3.4 mM). Because pHe decreased in response to the addition of lactic acid (Fig. 4b), we next examined whether lactic acid addition can decrease sperm intracellular pH (pHi). When pHrodo-loaded sperm were exposed to different concentrations of L-lactic acid, sperm pHi and pHe were concurrently decreased (Fig. 4b). On the other hand, intracellular ATP levels were not affected by the addition of L-lactic acid (Fig. 4c), which indicated that the lactic acid-induced sperm quiescence is not due to the depletion of intracellular ATP. Furthermore, a direct pHi measurement of SST-stored sperm, which was enabled by intra-vaginal insemination of pHrodo-loaded sperm, clearly demonstrated that intracellular acidosis of the resident sperm occurred (Fig. 4d,e,g, pH 6.2 ± 0.1, means ± SE, n = 29).

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