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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: These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility.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

The expression pattern of CCNYL1 in mice.(A) Ccnyl1 and Ccny mRNA levels were measured in different tissues of adult WT mice (n = 4). (B) Ccnyl1 and Ccny mRNA levels were measured in testes of ICR and BABL/c mouse lines (n = 4 per group). (C) Both Ccnyl1 and Ccny mRNA levels were measured in testes of WT mice of different age (n = 4). (D) CCNYL1 protein levels were measured in different tissues of adult WT mice, and in testes of WT mice of different ages. β-actin served as the protein loading control. (E) Immunolabeling of CCNYL1 (red) during the spermatogenic cycle of testicular sections. Nuclei were labeled with DAPI (blue). R: round spermatids (white arrows); E: elongated spermatids (yellow arrows); spermatozoa with shed residual bodies (asterisks). Scale bar for left and middle panels: 50 μm, scale bar for right panel: 25 μm. (F) CCNYL1 protein expression was measured in membrane extracts (designated as M) and cytoplasmic extracts (designated as C) isolated from testicular or germ cells of WT and Ccnyl1-/- mice. N-cadherin served as the membrane protein-loading control, whereas β-actin served as the cytoplasmic protein loading control. Data in bar graphs are presented as mean ± SEM.
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pgen.1005485.g001: The expression pattern of CCNYL1 in mice.(A) Ccnyl1 and Ccny mRNA levels were measured in different tissues of adult WT mice (n = 4). (B) Ccnyl1 and Ccny mRNA levels were measured in testes of ICR and BABL/c mouse lines (n = 4 per group). (C) Both Ccnyl1 and Ccny mRNA levels were measured in testes of WT mice of different age (n = 4). (D) CCNYL1 protein levels were measured in different tissues of adult WT mice, and in testes of WT mice of different ages. β-actin served as the protein loading control. (E) Immunolabeling of CCNYL1 (red) during the spermatogenic cycle of testicular sections. Nuclei were labeled with DAPI (blue). R: round spermatids (white arrows); E: elongated spermatids (yellow arrows); spermatozoa with shed residual bodies (asterisks). Scale bar for left and middle panels: 50 μm, scale bar for right panel: 25 μm. (F) CCNYL1 protein expression was measured in membrane extracts (designated as M) and cytoplasmic extracts (designated as C) isolated from testicular or germ cells of WT and Ccnyl1-/- mice. N-cadherin served as the membrane protein-loading control, whereas β-actin served as the cytoplasmic protein loading control. Data in bar graphs are presented as mean ± SEM.

Mentions: Real-time PCR results showed that Ccnyl1 was expressed at strikingly high levels in the testis, and was about seven times higher than that of its homologue, Ccny (Fig 1A), and similar results were also obtained from mice of other backgrounds, such as ICR (Fig 1B). Unlike Ccny, which had a relative stable expression pattern, Ccnyl1 was upregulated from the age of three weeks, and gradually reached a plateau at stages of sexual maturity (Fig 1C), which indicated its role in later stages of spermatogenesis [19]. The expression of Ccnyl1 was confirmed at the protein levels (Fig 1D). To gain more detailed insight into the expression of Ccnyl1, we isolated the germinal cell populations from seminiferous tubules of the testis from sexually matured mice by FACS (S1A Fig) [20]. The mRNA expression results showed that Ccnyl1 began to be highly expressed in meiotic cells, and gradually increased until the stages with round spermatids. A dramatic decrease of Ccnyl1 mRNA levels was found in elongating/elongated spermatids (S1B Fig). Both Ccnyl1 mRNA and protein levels were negligible in spermatogonia (Fig 1E, S1B and S1C Fig). The immunolabeling and immunoblot results showed that CCNYL1 protein was highly expressed on the membrane of meiotic spermatocytes (Fig 1E white arrows, and S1C Fig). Contrary to the mRNA expression levels, CCNYL1 protein was still abundant on the membrane of the elongating/elongated spermatids (Fig 1E, yellow arrows), but was absent from spermatozoa when the residual body was shed (Fig 1E, asterisks). To confirm the subcellular localization of CCNYL1, we isolated the membrane and cytoplasmic proteins, and demonstrated the expression of CCNYL1 on the membrane proteins fraction (Fig 1F). Taken together, these data provide evidence that CCNYL1 might play a critical role in spermatogenesis.


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)

The expression pattern of CCNYL1 in mice.(A) Ccnyl1 and Ccny mRNA levels were measured in different tissues of adult WT mice (n = 4). (B) Ccnyl1 and Ccny mRNA levels were measured in testes of ICR and BABL/c mouse lines (n = 4 per group). (C) Both Ccnyl1 and Ccny mRNA levels were measured in testes of WT mice of different age (n = 4). (D) CCNYL1 protein levels were measured in different tissues of adult WT mice, and in testes of WT mice of different ages. β-actin served as the protein loading control. (E) Immunolabeling of CCNYL1 (red) during the spermatogenic cycle of testicular sections. Nuclei were labeled with DAPI (blue). R: round spermatids (white arrows); E: elongated spermatids (yellow arrows); spermatozoa with shed residual bodies (asterisks). Scale bar for left and middle panels: 50 μm, scale bar for right panel: 25 μm. (F) CCNYL1 protein expression was measured in membrane extracts (designated as M) and cytoplasmic extracts (designated as C) isolated from testicular or germ cells of WT and Ccnyl1-/- mice. N-cadherin served as the membrane protein-loading control, whereas β-actin served as the cytoplasmic protein loading control. Data in bar graphs are presented as mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549061&req=5

pgen.1005485.g001: The expression pattern of CCNYL1 in mice.(A) Ccnyl1 and Ccny mRNA levels were measured in different tissues of adult WT mice (n = 4). (B) Ccnyl1 and Ccny mRNA levels were measured in testes of ICR and BABL/c mouse lines (n = 4 per group). (C) Both Ccnyl1 and Ccny mRNA levels were measured in testes of WT mice of different age (n = 4). (D) CCNYL1 protein levels were measured in different tissues of adult WT mice, and in testes of WT mice of different ages. β-actin served as the protein loading control. (E) Immunolabeling of CCNYL1 (red) during the spermatogenic cycle of testicular sections. Nuclei were labeled with DAPI (blue). R: round spermatids (white arrows); E: elongated spermatids (yellow arrows); spermatozoa with shed residual bodies (asterisks). Scale bar for left and middle panels: 50 μm, scale bar for right panel: 25 μm. (F) CCNYL1 protein expression was measured in membrane extracts (designated as M) and cytoplasmic extracts (designated as C) isolated from testicular or germ cells of WT and Ccnyl1-/- mice. N-cadherin served as the membrane protein-loading control, whereas β-actin served as the cytoplasmic protein loading control. Data in bar graphs are presented as mean ± SEM.
Mentions: Real-time PCR results showed that Ccnyl1 was expressed at strikingly high levels in the testis, and was about seven times higher than that of its homologue, Ccny (Fig 1A), and similar results were also obtained from mice of other backgrounds, such as ICR (Fig 1B). Unlike Ccny, which had a relative stable expression pattern, Ccnyl1 was upregulated from the age of three weeks, and gradually reached a plateau at stages of sexual maturity (Fig 1C), which indicated its role in later stages of spermatogenesis [19]. The expression of Ccnyl1 was confirmed at the protein levels (Fig 1D). To gain more detailed insight into the expression of Ccnyl1, we isolated the germinal cell populations from seminiferous tubules of the testis from sexually matured mice by FACS (S1A Fig) [20]. The mRNA expression results showed that Ccnyl1 began to be highly expressed in meiotic cells, and gradually increased until the stages with round spermatids. A dramatic decrease of Ccnyl1 mRNA levels was found in elongating/elongated spermatids (S1B Fig). Both Ccnyl1 mRNA and protein levels were negligible in spermatogonia (Fig 1E, S1B and S1C Fig). The immunolabeling and immunoblot results showed that CCNYL1 protein was highly expressed on the membrane of meiotic spermatocytes (Fig 1E white arrows, and S1C Fig). Contrary to the mRNA expression levels, CCNYL1 protein was still abundant on the membrane of the elongating/elongated spermatids (Fig 1E, yellow arrows), but was absent from spermatozoa when the residual body was shed (Fig 1E, asterisks). To confirm the subcellular localization of CCNYL1, we isolated the membrane and cytoplasmic proteins, and demonstrated the expression of CCNYL1 on the membrane proteins fraction (Fig 1F). Taken together, these data provide evidence that CCNYL1 might play a critical role in spermatogenesis.

Bottom Line: These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility.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