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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 were subjected to hypoxic conditions.(a) Differential gene expression between the non-SST cells and SST cells. Box plots represent log10(FPKM + 1) gene expression levels for the functional gene sets related to oncogene, pyruvate dehydrogenase kinase and lactate dehydrogenase (PDK&LDH), glycolysis, and glucose transporter (GLUT) in non-SST cells (Non_SSTs) and SST cells (SSTs). Colored lines between the gene expression values in Non_SSTs and SSTs represent log2-fold changes (LogFC), with values indicated on the color scale to the left. n, numbers of genes in the functional gene sets; Z and p, Z scores and p-values, respectively, calculated by parametric analysis of gene set enrichment based on the logFC between Non_SSTs and SSTs. (b,c) Immunohistochemical detection of hypoxic cells. UVJ tissues isolated from birds injected with HypoxyprobeTM. (b) or saline (c) were stained with anti-pimonidazole antibody (green). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Arrows and double arrows indicate SSTs and surface epithelium, respectively. Scale bar = 100 μm. (d,e) Mitochondrial activities of SSTs and surface epithelium assessed by JC-1 fluorescence in the presence (e) or absence (d) of an uncoupler. Surface epithelium displayed high mitochondrial activities (double arrow in panel (d)) while low mitochondrial activities were exhibited by SSTs (arrow in panel (d)). On the other hand, differences in mitochondrial activities between surface epithelium (double arrow in panel (e)) and SSTs (arrow in panels (e)) were absent in the presence of an uncoupler (panel (e)). Scale bar = 100 μm. (f) FI ratio (590 nm/530 nm) in the presence (hatched bars) or absence (solid bars) of an uncoupler. FI ratio indicated mitochondrial activity. Mitochondrial activities of SSTs were significantly lower than those of surface epithelium (Non-SSTs). Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.0004).
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f3: Sperm storage tubules were subjected to hypoxic conditions.(a) Differential gene expression between the non-SST cells and SST cells. Box plots represent log10(FPKM + 1) gene expression levels for the functional gene sets related to oncogene, pyruvate dehydrogenase kinase and lactate dehydrogenase (PDK&LDH), glycolysis, and glucose transporter (GLUT) in non-SST cells (Non_SSTs) and SST cells (SSTs). Colored lines between the gene expression values in Non_SSTs and SSTs represent log2-fold changes (LogFC), with values indicated on the color scale to the left. n, numbers of genes in the functional gene sets; Z and p, Z scores and p-values, respectively, calculated by parametric analysis of gene set enrichment based on the logFC between Non_SSTs and SSTs. (b,c) Immunohistochemical detection of hypoxic cells. UVJ tissues isolated from birds injected with HypoxyprobeTM. (b) or saline (c) were stained with anti-pimonidazole antibody (green). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Arrows and double arrows indicate SSTs and surface epithelium, respectively. Scale bar = 100 μm. (d,e) Mitochondrial activities of SSTs and surface epithelium assessed by JC-1 fluorescence in the presence (e) or absence (d) of an uncoupler. Surface epithelium displayed high mitochondrial activities (double arrow in panel (d)) while low mitochondrial activities were exhibited by SSTs (arrow in panel (d)). On the other hand, differences in mitochondrial activities between surface epithelium (double arrow in panel (e)) and SSTs (arrow in panels (e)) were absent in the presence of an uncoupler (panel (e)). Scale bar = 100 μm. (f) FI ratio (590 nm/530 nm) in the presence (hatched bars) or absence (solid bars) of an uncoupler. FI ratio indicated mitochondrial activity. Mitochondrial activities of SSTs were significantly lower than those of surface epithelium (Non-SSTs). Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.0004).

Mentions: Next, the gene expression profile related to glycolysis was assessed using RNA-seq to infer the mechanism for the massive production of lactic acid. The data set obtained in this study has been deposited in the DDBJ Sequence Read Archive (http://trace.ddbj.nig.ac.jp/dra/index.html) under accession number DRA003919. Unexpectedly, the expression levels of key glycolytic enzymes, such as hexokinase, phosphofructokinase, and pyruvate kinase, as well as several glucose transporters were not significantly higher in SSTs than those in non-SST cells, suggesting that enhanced glycolysis is not the primary mechanism in the mass synthesis of lactic acid in the SSTs (Fig. 3a). In accord with this, a KEGG PATHWAY mapping analysis (http://www.genome.jp/kegg/pathway.html) with the SST transcriptome dataset revealed no sign of enhanced glycolysis. Analysis of differential gene expression profiles with several sets of gene ontology (GO) between SST cells and non-SST cells failed to uncover molecular evidence for the mass production of lactic acid in the SSTs (Supplementary Table S1 online). In contrast, we found a dramatic increase in the expression of c-Fos and c-Jun in SSTs compared to non-SST cells (Fig. 3a). As it is known that these oncogenes are upregulated in highly proliferating cells22 and in cancer cells under hypoxic conditions23, and hypoxia has been shown to be an alternative factor promoting lactic acid production in cells24, we hypothesized that in physiological condition, SST cells are subjected to hypoxia. To test this hypothesis, HypoxyprobeTM was used to detect hypoxic cells and tissues through immunohistochemistry. We found that the intensity of the hypoxic signal was much greater in SST cells than non-SST cells (Fig. 3b,c). In addition, a marked decrease in mitochondrial activity within SST cells supported the idea that oxygen is limited in SSTs (Fig. 3d,e,f). From these results, we concluded that hypoxia is the likely cause for the accumulation of lactic acid in SSTs.


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 were subjected to hypoxic conditions.(a) Differential gene expression between the non-SST cells and SST cells. Box plots represent log10(FPKM + 1) gene expression levels for the functional gene sets related to oncogene, pyruvate dehydrogenase kinase and lactate dehydrogenase (PDK&LDH), glycolysis, and glucose transporter (GLUT) in non-SST cells (Non_SSTs) and SST cells (SSTs). Colored lines between the gene expression values in Non_SSTs and SSTs represent log2-fold changes (LogFC), with values indicated on the color scale to the left. n, numbers of genes in the functional gene sets; Z and p, Z scores and p-values, respectively, calculated by parametric analysis of gene set enrichment based on the logFC between Non_SSTs and SSTs. (b,c) Immunohistochemical detection of hypoxic cells. UVJ tissues isolated from birds injected with HypoxyprobeTM. (b) or saline (c) were stained with anti-pimonidazole antibody (green). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Arrows and double arrows indicate SSTs and surface epithelium, respectively. Scale bar = 100 μm. (d,e) Mitochondrial activities of SSTs and surface epithelium assessed by JC-1 fluorescence in the presence (e) or absence (d) of an uncoupler. Surface epithelium displayed high mitochondrial activities (double arrow in panel (d)) while low mitochondrial activities were exhibited by SSTs (arrow in panel (d)). On the other hand, differences in mitochondrial activities between surface epithelium (double arrow in panel (e)) and SSTs (arrow in panels (e)) were absent in the presence of an uncoupler (panel (e)). Scale bar = 100 μm. (f) FI ratio (590 nm/530 nm) in the presence (hatched bars) or absence (solid bars) of an uncoupler. FI ratio indicated mitochondrial activity. Mitochondrial activities of SSTs were significantly lower than those of surface epithelium (Non-SSTs). Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.0004).
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Related In: Results  -  Collection

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Show All Figures
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f3: Sperm storage tubules were subjected to hypoxic conditions.(a) Differential gene expression between the non-SST cells and SST cells. Box plots represent log10(FPKM + 1) gene expression levels for the functional gene sets related to oncogene, pyruvate dehydrogenase kinase and lactate dehydrogenase (PDK&LDH), glycolysis, and glucose transporter (GLUT) in non-SST cells (Non_SSTs) and SST cells (SSTs). Colored lines between the gene expression values in Non_SSTs and SSTs represent log2-fold changes (LogFC), with values indicated on the color scale to the left. n, numbers of genes in the functional gene sets; Z and p, Z scores and p-values, respectively, calculated by parametric analysis of gene set enrichment based on the logFC between Non_SSTs and SSTs. (b,c) Immunohistochemical detection of hypoxic cells. UVJ tissues isolated from birds injected with HypoxyprobeTM. (b) or saline (c) were stained with anti-pimonidazole antibody (green). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Arrows and double arrows indicate SSTs and surface epithelium, respectively. Scale bar = 100 μm. (d,e) Mitochondrial activities of SSTs and surface epithelium assessed by JC-1 fluorescence in the presence (e) or absence (d) of an uncoupler. Surface epithelium displayed high mitochondrial activities (double arrow in panel (d)) while low mitochondrial activities were exhibited by SSTs (arrow in panel (d)). On the other hand, differences in mitochondrial activities between surface epithelium (double arrow in panel (e)) and SSTs (arrow in panels (e)) were absent in the presence of an uncoupler (panel (e)). Scale bar = 100 μm. (f) FI ratio (590 nm/530 nm) in the presence (hatched bars) or absence (solid bars) of an uncoupler. FI ratio indicated mitochondrial activity. Mitochondrial activities of SSTs were significantly lower than those of surface epithelium (Non-SSTs). Values shown are means ± SEM of three independent experiments. Different letters denote significant differences (P < 0.0004).
Mentions: Next, the gene expression profile related to glycolysis was assessed using RNA-seq to infer the mechanism for the massive production of lactic acid. The data set obtained in this study has been deposited in the DDBJ Sequence Read Archive (http://trace.ddbj.nig.ac.jp/dra/index.html) under accession number DRA003919. Unexpectedly, the expression levels of key glycolytic enzymes, such as hexokinase, phosphofructokinase, and pyruvate kinase, as well as several glucose transporters were not significantly higher in SSTs than those in non-SST cells, suggesting that enhanced glycolysis is not the primary mechanism in the mass synthesis of lactic acid in the SSTs (Fig. 3a). In accord with this, a KEGG PATHWAY mapping analysis (http://www.genome.jp/kegg/pathway.html) with the SST transcriptome dataset revealed no sign of enhanced glycolysis. Analysis of differential gene expression profiles with several sets of gene ontology (GO) between SST cells and non-SST cells failed to uncover molecular evidence for the mass production of lactic acid in the SSTs (Supplementary Table S1 online). In contrast, we found a dramatic increase in the expression of c-Fos and c-Jun in SSTs compared to non-SST cells (Fig. 3a). As it is known that these oncogenes are upregulated in highly proliferating cells22 and in cancer cells under hypoxic conditions23, and hypoxia has been shown to be an alternative factor promoting lactic acid production in cells24, we hypothesized that in physiological condition, SST cells are subjected to hypoxia. To test this hypothesis, HypoxyprobeTM was used to detect hypoxic cells and tissues through immunohistochemistry. We found that the intensity of the hypoxic signal was much greater in SST cells than non-SST cells (Fig. 3b,c). In addition, a marked decrease in mitochondrial activity within SST cells supported the idea that oxygen is limited in SSTs (Fig. 3d,e,f). From these results, we concluded that hypoxia is the likely cause for the accumulation of lactic acid in SSTs.

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