| Cyclin proteins are critical for the regulation of cell cycle process. Nowadays, many Cyclin proteins have been identified. The common character of these proteins is that all of them contained a conserved domain which is designated as Cyclin Box. In general, Cyclins interact with the Cyclin dependent kinases (Cdks), and then play a role by activating the kinase activity of Cdks. Though firstly identified in the regula-tion of Cell cycle progression, Cyclin/Cdk partners are also involved in regulating various celluar activities beyond cell cycle regulation.Cyclin Y-like 1 (Ccnyll) is a newly identified member of the cyclins family, and showed high similarity to Cyclin Y (Ccny). However, the function of Ccnyll is poorly characterized in any organisms. Here we found that Ccnyll was most abundantly ex-pressed in the testis of mice, whereas not in other tissues. Since Ccnyll and Ccny are homologue proteins, and show high similarity, we also measured the expression of Ccny in testis of WT mice, and found that Ccnyll have an overwhelming high expres-sion level towards Ccny. We also measured the expression of Ccnyll and Ccny in tes-tis of mice with different ages, and found that Ccnyll began to be strikingly upregu-lated from the age of three weeks, and gradually reached a plateau at stages of sexual maturity, while the expression of Ccny was relatively stable, and with little changes. Through immunostaining of testis sections and FACS analysis of different populations of germinal cells, we found Ccnyll was mainly expressed on the plasma membrane of spermatocytes and spermatids. All of these data indicated that Ccnyll might play an important role in spermatogenesis.To identify the function of Ccnyll, mice with Ccnyll knockout were employed. We observed no difference on the phenotype in Ccnyll-/-mice compared with their lit-termates. However, male Ccnyll-/-mice were infertile, but not the females. We also observed the fertility of Ccny knockout mice, in contrast, both male and female Ccny-/-mice presented normal fertility, indicating that Ccnyll, but not Ccny, was es-sential for male fertility. Male Ccnyll-/-mice showed normal body weight, reproduc-tive organ weights, serum testosterone and follicle-stimulating hormone levels. We also observed the morphology and structure of seminiferous tubules, cell counts and ratios of different populations of germinal cells, the expression of marker genes of the spermatocytes, and the sperm counts from both caput and cauda epididymidis, but no abnormality was found. These results demonstrate that Ccnyll have no effect on the development of male reproductive organs, the hormone levels, and the sperm count.To elucidate the defects underlying the sterility, we then measured the motility of sperms by computer-assisted semen analysis (CASA). The motility was greatly im-paired in Ccnyll-/-mice. We firstly examined the morphology of sperms under phase contrast microscope, many sperms collected from Ccnyll-/-mice showed bent head wrapped around by the neck. Almost all sperms of Ccnyll-/-mice displayed thinning of the annulus region, and about half of them also showed a sharply bend at this re-gion. In addition, the analysis with differential interference contrast microscope (DIC) showed that sperms collected from Ccnyll-/-mice were lack of cytoplasmic droplets. Though Ccnyll-/-spermatozoa showed several defects, their capacitation ability was intact. However, in vitro fertilization ability was greatly impaired in Ccnyll-/-mice. These data indicated that the structural defects and impaired motility of Ccnyll-/-spermatozoa might be responsible for infertility.To further characterize the morphological and motile abnormalities, we performed transmission electron microscopy (TEM) to analyze sperms. Ccnyll-/-spermatozoa displayed normal head, but bent heads and necks that were wrapped by the contents resembling the shedding cytoplasm, the thinning of the annulus region with mito-chondria detached from the mitochondria, and a breakage occurred at the thinning re-gion. Since Ccnyll-/-spermatozoa had normal mitochondrial structure and arrange-ment, and normal protein levels of mitochondrial markers such as Cytochrome C and Cytochrome c oxidase subunit 4, the thinning can not be ascribed to the defect of mi-tochondria. Furthermore, immunostaining showed that the microtubules and actin filaments were scattered out of the principle piece, which was further elucidated by the TEM. We speculated that defects of Ccnyll-/-spermatozoa might be caused by the abnormality of cytoskeleton. a-tubulin was equally expressed between WT and Ccnyll-/-spermatozoa, whereas the β-actin levels were significantly increased in Ccnyll-/-spermatozoa compared to that of WT mice. To further explain the increased β-actin levels in Ccnyll-/-spermatozoa, we measured key molecules involved in actin polymerization and depolymerization in testis, compared the F-actin and G-actin rati-os in both testis and spermatozoa, and further measured the activity of RhoA/Racl/Cdc42, which involved in actin cytoskeleton rearrangement, but no sig-nificant changes were observed. These lines of evidence indicate that the increased β-actin levels in Ccnyll-/-spermatozoa might be ascribed to the incomplete shedding of cytoplasmic contents rather than per se cytoskeleton defects, and the scattered mi- crotubules and actin filaments might be the result of the breakage of the thinning an-nulus region.To elucidate the mechanism of Ccnyll in spermatogenesis, we first screened the targets of Ccnyll by cDNA microarray analysis. However, there were little differences and none of them seemed to be particular interesting, indicating that Ccnyll might not participate in transcription regulation. For the homolog of Ccnyll, Ccny was involved in the regulation of canonical Wnt signaling pathway, we also screened this pathway, but gained nothing. Additionally, we screened the knockout mouse models with phe-notypes similar to that of Ccnyll-/-mice from the literature. Interestingly, spermato-zoa collected from Cdkl6-/-mice displayed a quite similar phenotype. Next, we measured the expression of Cdk16 in the testis. The mRNA levels of Cdk16 in Ccnyll-/-testis was similar to that of WT mice. However, when we checked the pro-tein levels, we found Cdk16 protein level was significantly decreased in Ccnyll-/-tes-tis. We then speculated that Ccnyll might have an interaction with Cdk16. Coim-munolabeling results showed that Ccnyll colocalized with Cdk16 in the testis, and this was further confirmed by both in vivo and in vitro Co-IP experiments.Since our data showed that Cdk16 protein level was decreased in Ccnyll-/-testis, and that Ccnyl 1 interacted with Cdk16 both in vivo and in vitro, we proposed that the recruitment of Cdk16 by Ccnyll might increase its stability or enhance the translation of Cdkl6 mRNA. We found that the interaction of Ccnyll and Cdk16 mutually in-crease their protein stabilities, which was supported by both the protein stability assay and the inhibition of protein degradation by MG132. In addition, we found that Cdkl6 shifted slightly lower than that of WT controls during protein gel electrophoresis analysis. This band-shift was eliminated by incubating the cell lysates with phospha-tase, implying that the presence of Ccnyll might relate to the phosphorylation status of Cdk16. We then fully analyzed the phosphorylation sites of Cdk16 by mass spec-trometry, and found that the N-terminal region of Cdk16 was highly phosphorylated. In total,22 phosphosites were identified,19 of which were determined with high con-fidence. Among those 19 sites, nine of them were newly identified, and had not been reported. They were S36, S64, S65, S89, S146, T175, T380, S391, and S478. Our re-sults showed that the phosphorylation levels of Cdkl6 were significantly increased at four sites (S36, S146, T175, and S480), while decreased at one site (S78), when co-overexpressed with Ccnyll. Thus, this explained the altered phosphorylation levels of Cdkl6 in the presence of Ccnyll. To examine whether the altered phosphorylation events of Cdk16 could affect the binding of Ccnyll and its kinase activity, we gener-ated site mutations on these phosphosites for Co-IP and kinase assay. We found that the phosphorylation modifications of the N-terminal region of Cdk16 were essential for the binding of Ccnyll. In addition, we investigated the kinase activity of Cdkl6 with the radio-label assay. Cdk16 had very low kinase activity when overexpressed alone, but the binding of Ccnyll strikingly increased the kinase activity of Cdk16. Among those newly identidied phosphorylation sites, S146 was critical for the bind-ing of Ccnyll and modulating the kinase activity of Cdk16. S146A and S146D muta-tions all led to decreased kinase activity of Cdkl6, and abolished binding of Ccnyll. When co-overexpressed with Ccnyll, the S146A and S146D mutations still displayed substantially decreased kinase activity, implying the importance of this site in modu-lating Cdk16 kinase activity. Taken together, the phosphorylation modifications on Cdk16 are necessary for the binding of Ccnyll, which results in both increased pro-tein stability and Cdk16 kinase activity.In summary, we firstly identify the function of Ccnyll,and reveal that the Ccnyll and Cdk16 protein complex is essential for spermatogenesis and male fertility. And speculate that, Ccnyll might be an ideal molecular target for male contraception in-tervention, as Ccnyll is specifically expressed in the testis, and Ccnyll-/-mice ap-peared healthy, with normal testosterone level and mating status, except the sterility. Finally, researches from mouse models continue to enrich our knowledge of the ge-netic basis of male infertility. These studies would be promising in translating the knowledge from animal models into strategies for diagnosis and treatment of male infertility. |
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