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CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Zi Z, Zhang Z, Li Q, An W, Zeng L, Gao D, Yang Y, Zhu X, Zeng R, Shum WW, Wu J - PLoS Genet. (2015)

Bottom Line: In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1.We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity.Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China.

ABSTRACT
Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

No MeSH data available.


Related in: MedlinePlus

Morphological defects of Ccnyl1-/- spermatozoa.(A) Phase Contrast images of spermatozoa collected from the testis, caput and cauda epididymidis of adult WT and Ccnyl1-/- mice. Ccnyl1-/- spermatozoa showed a thinning of annulus (white arrows) and a bent head wrapped around the neck (red arrows). Scale bar: 20μm. The ratios of defects are summarized in the histograms below (mice: n≥ 3 animals per group). Data are presented as mean ± SEM. (B) TEM images of spermatozoa collected from the cauda epididymidis of adult WT and Ccnyl1-/- mice. MP: Middle Piece; PP: Principal Piece. The annulus structure (black arrows) closely linked the MP and PP in WT spermatozoa, but was distant from the MP as the thinning of the annulus region in Ccnyl1-/- spermatozoa. Microtubules (green arrows) were dispersed out of the PP in Ccnyl1-/- spermatozoa. Two Ccnyl1-/- spermatozoa with bent heads wrapped by cytoplasmic contents are shown (blue arrows). Scale bar for the top and middle panels: 0.5 μm, scale bar for the bottom panels: 1 μm. (C and D) Immuno-labeling of α-tubulin (C) and F-actin (D) of spermatozoa collected from cauda epididymidis of adult WT and Ccnyl1-/- mice. Microtubules (yellow arrows) and F-actin (pink arrows) were extruded from of this region in spermatozoa of Ccnyl1-/- mice, scale bar: 5 μm.
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pgen.1005485.g003: Morphological defects of Ccnyl1-/- spermatozoa.(A) Phase Contrast images of spermatozoa collected from the testis, caput and cauda epididymidis of adult WT and Ccnyl1-/- mice. Ccnyl1-/- spermatozoa showed a thinning of annulus (white arrows) and a bent head wrapped around the neck (red arrows). Scale bar: 20μm. The ratios of defects are summarized in the histograms below (mice: n≥ 3 animals per group). Data are presented as mean ± SEM. (B) TEM images of spermatozoa collected from the cauda epididymidis of adult WT and Ccnyl1-/- mice. MP: Middle Piece; PP: Principal Piece. The annulus structure (black arrows) closely linked the MP and PP in WT spermatozoa, but was distant from the MP as the thinning of the annulus region in Ccnyl1-/- spermatozoa. Microtubules (green arrows) were dispersed out of the PP in Ccnyl1-/- spermatozoa. Two Ccnyl1-/- spermatozoa with bent heads wrapped by cytoplasmic contents are shown (blue arrows). Scale bar for the top and middle panels: 0.5 μm, scale bar for the bottom panels: 1 μm. (C and D) Immuno-labeling of α-tubulin (C) and F-actin (D) of spermatozoa collected from cauda epididymidis of adult WT and Ccnyl1-/- mice. Microtubules (yellow arrows) and F-actin (pink arrows) were extruded from of this region in spermatozoa of Ccnyl1-/- mice, scale bar: 5 μm.

Mentions: To find the cause of the impaired sperm motility of Ccnyl1 knockout mice, we examined their sperm morphology. Almost all spermatozoa isolated from the caput and cauda epididymidis of Ccnyl1-/- mice displayed thinning of the annulus region (Fig 3A). About half the cauda spermatozoa displayed a sharp bend at the annulus region. Moreover, many sperm head were bent and wrapped around the neck (Fig 3A). In addition, the analysis with differential interference contrast microscope revealed that spermatozoa from the cauda epididymidis of Ccnyl1-/- mice lacked of cytoplasmic droplets (S5A Fig). This abnormal frequency was less evident in spermatozoa collected from more proximal epididymal regions (Fig 3A, bottom panel, and S5A Fig). Only a small percentage of Ccnyl1-/- spermatozoa showed the morphological phenotype in the testis (Fig 3A). In addition, high expression of CCNYL1 and CDK16 was found in the testicular spermatocytes and spermatids, rather than in the epididymis, and also CDK16 protein levels were no lower in the epididymis of Ccnyl1-/- mice than in that of WT mice (S4C and S4G Fig). Moreover, there were no significant changes in epididymal Ccnyl1 expression in mice of different ages (S4F Fig). Thus, we concluded that it was the lack of Ccnyl1 in the testis and the subsequently decreased CDK16 protein levels that led to the abnormality of sperms. The gradually increased morphological defects of sperms might be due to the mechanical stress during the transition through the epididymis or the onset of motility of the sperms themselves.


CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Zi Z, Zhang Z, Li Q, An W, Zeng L, Gao D, Yang Y, Zhu X, Zeng R, Shum WW, Wu J - PLoS Genet. (2015)

Morphological defects of Ccnyl1-/- spermatozoa.(A) Phase Contrast images of spermatozoa collected from the testis, caput and cauda epididymidis of adult WT and Ccnyl1-/- mice. Ccnyl1-/- spermatozoa showed a thinning of annulus (white arrows) and a bent head wrapped around the neck (red arrows). Scale bar: 20μm. The ratios of defects are summarized in the histograms below (mice: n≥ 3 animals per group). Data are presented as mean ± SEM. (B) TEM images of spermatozoa collected from the cauda epididymidis of adult WT and Ccnyl1-/- mice. MP: Middle Piece; PP: Principal Piece. The annulus structure (black arrows) closely linked the MP and PP in WT spermatozoa, but was distant from the MP as the thinning of the annulus region in Ccnyl1-/- spermatozoa. Microtubules (green arrows) were dispersed out of the PP in Ccnyl1-/- spermatozoa. Two Ccnyl1-/- spermatozoa with bent heads wrapped by cytoplasmic contents are shown (blue arrows). Scale bar for the top and middle panels: 0.5 μm, scale bar for the bottom panels: 1 μm. (C and D) Immuno-labeling of α-tubulin (C) and F-actin (D) of spermatozoa collected from cauda epididymidis of adult WT and Ccnyl1-/- mice. Microtubules (yellow arrows) and F-actin (pink arrows) were extruded from of this region in spermatozoa of Ccnyl1-/- mice, scale bar: 5 μm.
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Related In: Results  -  Collection

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Show All Figures
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pgen.1005485.g003: Morphological defects of Ccnyl1-/- spermatozoa.(A) Phase Contrast images of spermatozoa collected from the testis, caput and cauda epididymidis of adult WT and Ccnyl1-/- mice. Ccnyl1-/- spermatozoa showed a thinning of annulus (white arrows) and a bent head wrapped around the neck (red arrows). Scale bar: 20μm. The ratios of defects are summarized in the histograms below (mice: n≥ 3 animals per group). Data are presented as mean ± SEM. (B) TEM images of spermatozoa collected from the cauda epididymidis of adult WT and Ccnyl1-/- mice. MP: Middle Piece; PP: Principal Piece. The annulus structure (black arrows) closely linked the MP and PP in WT spermatozoa, but was distant from the MP as the thinning of the annulus region in Ccnyl1-/- spermatozoa. Microtubules (green arrows) were dispersed out of the PP in Ccnyl1-/- spermatozoa. Two Ccnyl1-/- spermatozoa with bent heads wrapped by cytoplasmic contents are shown (blue arrows). Scale bar for the top and middle panels: 0.5 μm, scale bar for the bottom panels: 1 μm. (C and D) Immuno-labeling of α-tubulin (C) and F-actin (D) of spermatozoa collected from cauda epididymidis of adult WT and Ccnyl1-/- mice. Microtubules (yellow arrows) and F-actin (pink arrows) were extruded from of this region in spermatozoa of Ccnyl1-/- mice, scale bar: 5 μm.
Mentions: To find the cause of the impaired sperm motility of Ccnyl1 knockout mice, we examined their sperm morphology. Almost all spermatozoa isolated from the caput and cauda epididymidis of Ccnyl1-/- mice displayed thinning of the annulus region (Fig 3A). About half the cauda spermatozoa displayed a sharp bend at the annulus region. Moreover, many sperm head were bent and wrapped around the neck (Fig 3A). In addition, the analysis with differential interference contrast microscope revealed that spermatozoa from the cauda epididymidis of Ccnyl1-/- mice lacked of cytoplasmic droplets (S5A Fig). This abnormal frequency was less evident in spermatozoa collected from more proximal epididymal regions (Fig 3A, bottom panel, and S5A Fig). Only a small percentage of Ccnyl1-/- spermatozoa showed the morphological phenotype in the testis (Fig 3A). In addition, high expression of CCNYL1 and CDK16 was found in the testicular spermatocytes and spermatids, rather than in the epididymis, and also CDK16 protein levels were no lower in the epididymis of Ccnyl1-/- mice than in that of WT mice (S4C and S4G Fig). Moreover, there were no significant changes in epididymal Ccnyl1 expression in mice of different ages (S4F Fig). Thus, we concluded that it was the lack of Ccnyl1 in the testis and the subsequently decreased CDK16 protein levels that led to the abnormality of sperms. The gradually increased morphological defects of sperms might be due to the mechanical stress during the transition through the epididymis or the onset of motility of the sperms themselves.

Bottom Line: In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1.We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity.Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China.

ABSTRACT
Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

No MeSH data available.


Related in: MedlinePlus