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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

Essential function of CCNYL1 for male fertility and sperm motility.(A) Measurement of CCNYL1 or CCNY protein expression in testes of WT, Ccnyl1-/-, and Ccny-/- mice, with β-actin serving as loading control. (B) Male fertility of adult WT, Ccnyl1-/- and Ccny-/- mice (n ≥ 6 per group). Pregnancy was counted in mating cages with male WT, Ccnyl1-/-, or Ccny-/- mice. (C) Number of spermatozoa in male WT, Ccnyl1-/-, and Ccny-/- mice collected from the caput and cauda epididymidis (n ≥ 6 per group). (D) HE staining of testicular sections from adult WT and Ccnyl1-/- mice. Scale bar: 100μm. (E-G) The percentage of total and progressive motility (E), velocity (F) and the motility distribution (G) of spermatozoa were measured by CASA (n = 5 animals per group). (H) In vitro fertilization assay. Oocytes were incubated with cauda epididymidal spermatozoa collected from WT or Ccnyl1-/- mice. The development of the oocytes was monitored in vitro. Data in bar graphs are presented as mean ± SEM. *P < 0.05; **P < 0.01.
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pgen.1005485.g002: Essential function of CCNYL1 for male fertility and sperm motility.(A) Measurement of CCNYL1 or CCNY protein expression in testes of WT, Ccnyl1-/-, and Ccny-/- mice, with β-actin serving as loading control. (B) Male fertility of adult WT, Ccnyl1-/- and Ccny-/- mice (n ≥ 6 per group). Pregnancy was counted in mating cages with male WT, Ccnyl1-/-, or Ccny-/- mice. (C) Number of spermatozoa in male WT, Ccnyl1-/-, and Ccny-/- mice collected from the caput and cauda epididymidis (n ≥ 6 per group). (D) HE staining of testicular sections from adult WT and Ccnyl1-/- mice. Scale bar: 100μm. (E-G) The percentage of total and progressive motility (E), velocity (F) and the motility distribution (G) of spermatozoa were measured by CASA (n = 5 animals per group). (H) In vitro fertilization assay. Oocytes were incubated with cauda epididymidal spermatozoa collected from WT or Ccnyl1-/- mice. The development of the oocytes was monitored in vitro. Data in bar graphs are presented as mean ± SEM. *P < 0.05; **P < 0.01.

Mentions: To determine the function of Ccnyl1, mice with the gene knockout were studied [21] (Fig 2A top panel, S2A Fig). We observed no difference in the phenotype of Ccnyl1-/- mice from that of their WT littermates. However, when Ccnyl1-/- males were mated with the wild type females, none of the females became pregnant (Fig 2B). In contrast, both male and female Ccny knockout mice (S2B Fig) and female Ccnyl1-/- mice presented were fertile (Fig 2B and S1 Table), indicating that Ccnyl1, but not Ccny, was essential for male fertility. Male Ccnyl1-/- mice showed normal body weight and reproductive organ weights (S3A and S3B Fig). The serum testosterone and follicle-stimulating hormone levels in Ccnyl1-/- mice were not abnormal (S3C and S3D Fig). To elucidate the defects underlying the sterility, we measured the number of spermatozoa obtained from the caput and cauda epididymidis, but observed no difference in the sperm counts between Ccnyl1-/- and WT mice in either region (Fig 2C). Furthermore, histological and FACS examination showed no overt effect of Ccnyl1 deficiency at any stages of germ cell development, which was also supported by similarity in genes specifically expressed by different germinal cell populations (Fig 2D, S3E and S3F Fig). The motility of cauda epididymidal spermatozoa by computer-assisted sperm analysis (CASA) revealed that the percentages of total and progressive motility were greatly reduced in Ccnyl1-/- mice (Fig 2E), and the progressive spermatozoa displayed significantly reduced velocity (Fig 2F) though a slightly increased beat frequency (S3G Fig). A higher percentage of spermatozoa from the Ccnyl1-/- mice were static or of low motility than that of WT mice (Fig 2G), whereas spermatozoa obtained from Ccny-/- mice showed sperm counts and sperm motility no different from those of WT mice (Fig 2C and 2E–2G). Though Ccnyl1-/- spermatozoa showed several defects, their capacitation ability was intact (S3H Fig). The ability of Ccnyl1-/- mouse spermatozoa to fertilize oocytes in vitro was greatly impaired; only six oocytes were fertilized, and two developed to blastocysts in vitro compared with WT controls in which 152 oocytes were fertilized and 71 blastocysts obtained in the same set of experiments (Fig 2H). These data indicated that the structural defects and impaired motility of Ccnyl1-/- spermatozoa might prevent their propulsion and entry into oocytes, and then lead to impaired fertilization. However, we could not exclude that other uncharacterized defects might also be involved.


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)

Essential function of CCNYL1 for male fertility and sperm motility.(A) Measurement of CCNYL1 or CCNY protein expression in testes of WT, Ccnyl1-/-, and Ccny-/- mice, with β-actin serving as loading control. (B) Male fertility of adult WT, Ccnyl1-/- and Ccny-/- mice (n ≥ 6 per group). Pregnancy was counted in mating cages with male WT, Ccnyl1-/-, or Ccny-/- mice. (C) Number of spermatozoa in male WT, Ccnyl1-/-, and Ccny-/- mice collected from the caput and cauda epididymidis (n ≥ 6 per group). (D) HE staining of testicular sections from adult WT and Ccnyl1-/- mice. Scale bar: 100μm. (E-G) The percentage of total and progressive motility (E), velocity (F) and the motility distribution (G) of spermatozoa were measured by CASA (n = 5 animals per group). (H) In vitro fertilization assay. Oocytes were incubated with cauda epididymidal spermatozoa collected from WT or Ccnyl1-/- mice. The development of the oocytes was monitored in vitro. Data in bar graphs are presented as mean ± SEM. *P < 0.05; **P < 0.01.
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Related In: Results  -  Collection

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

pgen.1005485.g002: Essential function of CCNYL1 for male fertility and sperm motility.(A) Measurement of CCNYL1 or CCNY protein expression in testes of WT, Ccnyl1-/-, and Ccny-/- mice, with β-actin serving as loading control. (B) Male fertility of adult WT, Ccnyl1-/- and Ccny-/- mice (n ≥ 6 per group). Pregnancy was counted in mating cages with male WT, Ccnyl1-/-, or Ccny-/- mice. (C) Number of spermatozoa in male WT, Ccnyl1-/-, and Ccny-/- mice collected from the caput and cauda epididymidis (n ≥ 6 per group). (D) HE staining of testicular sections from adult WT and Ccnyl1-/- mice. Scale bar: 100μm. (E-G) The percentage of total and progressive motility (E), velocity (F) and the motility distribution (G) of spermatozoa were measured by CASA (n = 5 animals per group). (H) In vitro fertilization assay. Oocytes were incubated with cauda epididymidal spermatozoa collected from WT or Ccnyl1-/- mice. The development of the oocytes was monitored in vitro. Data in bar graphs are presented as mean ± SEM. *P < 0.05; **P < 0.01.
Mentions: To determine the function of Ccnyl1, mice with the gene knockout were studied [21] (Fig 2A top panel, S2A Fig). We observed no difference in the phenotype of Ccnyl1-/- mice from that of their WT littermates. However, when Ccnyl1-/- males were mated with the wild type females, none of the females became pregnant (Fig 2B). In contrast, both male and female Ccny knockout mice (S2B Fig) and female Ccnyl1-/- mice presented were fertile (Fig 2B and S1 Table), indicating that Ccnyl1, but not Ccny, was essential for male fertility. Male Ccnyl1-/- mice showed normal body weight and reproductive organ weights (S3A and S3B Fig). The serum testosterone and follicle-stimulating hormone levels in Ccnyl1-/- mice were not abnormal (S3C and S3D Fig). To elucidate the defects underlying the sterility, we measured the number of spermatozoa obtained from the caput and cauda epididymidis, but observed no difference in the sperm counts between Ccnyl1-/- and WT mice in either region (Fig 2C). Furthermore, histological and FACS examination showed no overt effect of Ccnyl1 deficiency at any stages of germ cell development, which was also supported by similarity in genes specifically expressed by different germinal cell populations (Fig 2D, S3E and S3F Fig). The motility of cauda epididymidal spermatozoa by computer-assisted sperm analysis (CASA) revealed that the percentages of total and progressive motility were greatly reduced in Ccnyl1-/- mice (Fig 2E), and the progressive spermatozoa displayed significantly reduced velocity (Fig 2F) though a slightly increased beat frequency (S3G Fig). A higher percentage of spermatozoa from the Ccnyl1-/- mice were static or of low motility than that of WT mice (Fig 2G), whereas spermatozoa obtained from Ccny-/- mice showed sperm counts and sperm motility no different from those of WT mice (Fig 2C and 2E–2G). Though Ccnyl1-/- spermatozoa showed several defects, their capacitation ability was intact (S3H Fig). The ability of Ccnyl1-/- mouse spermatozoa to fertilize oocytes in vitro was greatly impaired; only six oocytes were fertilized, and two developed to blastocysts in vitro compared with WT controls in which 152 oocytes were fertilized and 71 blastocysts obtained in the same set of experiments (Fig 2H). These data indicated that the structural defects and impaired motility of Ccnyl1-/- spermatozoa might prevent their propulsion and entry into oocytes, and then lead to impaired fertilization. However, we could not exclude that other uncharacterized defects might also be involved.

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