of the N-terminal domains of

common phospholipid-binding activit

Complementary Critical Functions of <i>Zfy1</i> and <i>Zfy2</i> in Mouse Spermatogenesis and Reproduction

<div><p>The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. <i>Zfy</i> was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, <i>Zfy</i> gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual <i>Zfy1</i> or <i>Zfy2</i> knockout (KO) mice and <i>Zfy1</i> and <i>Zfy2</i> double knockout (<i>Zfy1/2</i>-DKO) mice. While individual <i>Zfy1</i> or <i>Zfy2-</i>KO mice did not show any significant phenotypic alterations in fertility, <i>Zfy1/2-</i>DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of <i>Zfy1/2-</i>DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that <i>Zfy1</i> and <i>Zfy2</i> have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure.</p></div

Sperm proteins downregulated in <i>Zfy1</i> and <i>Zfy2</i> double knockout (<i>Zfy1/2</i>-DKO) mice.

<p>(A) Mass spectrometric protein profiling of WT and <i>Zfy1/2</i>-DKO sperm. (B) Western blot analysis of sperm proteins extracted from cauda epididymis. α/β tubulin and β-actin were used as internal controls. (C) Scheme of proposed function of <i>Zfy</i>.</p

Histology analysis of testis and epididymis.

<p>(A to D) Gross appearance of the testis and epididymis. WT (A), <i>Zfy-1</i> knockout (KO) (B), <i>Zfy2</i>-KO (C), <i>Zfy1</i> and <i>Zfy2</i> double knockout (<i>Zfy1/2</i>-DKO) (D). (E) Testis weight of WT, <i>Zfy1</i>-KO and 2-KO, and 1/2-DKO mice. Error bars: S.D., n = 3. n.s.: not significant. (F) Sperm counts in the testis. Error bars: S.D., n = 3. (G) Sperm counts in the epididymis. Error bars: S.D., n = 3. (H) HE and periodic acid-Schiff (PAS)-haematoxylin staining of the testis and the cauda epididymis section of WT, <i>Zfy1</i>-KO, <i>Zfy2</i>-KO, and <i>Zfy1/2</i>-DKO mice. Roman numerals indicate the stages of the seminiferous tubules.</p

Strategy for and efficiency of generation of <i>Zfy1</i> knockout (KO) mice, <i>Zfy2</i>-KO mice, and <i>Zfy1</i> and <i>Zfy2</i> double knockout (<i>Zfy1/2</i>-DKO) mice, and the corresponding fertility of the mice.

<p>(A) Target sequence of <i>Zfy1</i> and <i>Zfy2</i> for guide RNA (gRNA). PAM; proto-spacer adjacent motif. (B) Strategy for microinjection and embryo transfer, and efficiency of generation of mutant mice. (C to F) Vaginal plug of WT (C), <i>Zfy1</i>-KO (D), <i>Zfy2</i>-KO (E), and <i>Zfy1/2</i>-DKO (F). (G) Pregnancy success rates of WT, <i>Zfy1</i>-KO, <i>Zfy</i>2-KO, and <i>Zfy</i>1/2-DKO mice were 100%, 100%, 60%, and 0%, respectively. (H) Average litter sizes of WT, <i>Zfy1</i>-KO, <i>Zfy</i>2-KO, and <i>Zfy</i>1/2-DKO mice were 10.7, 10.3, 7, and 0, respectively. Error bars; S.D., n = 10, ** <i>p</i> < 0.01 compared with WT (Student’s <i>t</i>-test).</p

Histology and motility analysis of sperm.

<p>(A) Morphology of sperm in WT, <i>Zfy1</i> knockout (KO), <i>Zfy2</i>-KO, and <i>Zfy1</i> and <i>Zfy2</i> double knockout (<i>Zfy1/2</i>-DKO). (B) Transmission electron microscope analysis of WT, <i>Zfy2</i>-KO, and <i>Zfy1/2</i>-DKO. Arrows: abnormal mitochondria. (C) Summary of sperm morphology. Sperm were counted as more than 100 using an optical microscope. Error bars: S.D., n <i>≥</i> 3, * <i>p</i> < 0.05, ** <i>p</i> < 0.01 compared with WT (Student’s <i>t</i>-test). Sperm were counted as more than 50 using an electron microscope (<i>Zfy1</i>-KO; no data). n = 1, *** <i>p</i> < 0.01 compared with WT (two-tailed Fisher’s exact test). (D) Immunostaining of sperm. Epididymal sperm were separated on the glass slide and examined by fluorescent microscopy. Histological observations showed morphological abnormalities of mutant sperm. Formation of the nucleus, acrosome, and mitochondrion of the sperm were examined with DAPI, peanut agglutinin (PNA), and MitoTracker (Mit), respectively. Arrows indicated abnormal mitochondria. (E) Sperm motility was analyzed using computer-assisted sperm analysis. The ratios of motile sperm in WT, <i>Zfy1-KO</i>, <i>Zfy2</i>-KO, and <i>Zfy1/2</i>-DKO mice were 85.6%, 83.5%, 70.0%, 1.0%, respectively. Error bars; S.E.M. (WT, <i>Zfy1</i>-KO, and <i>Zfy2</i>-KO; n = 4, <i>Zfy1/2</i>-DKO; n = 1). ** <i>p</i> < 0.01 compared with WT (Student’s <i>t</i>-test).</p

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression

Summary: Histone deacetylases (HDACs) are promising targets for cancer therapy, although their individual actions remain incompletely understood. Here, we identify a role for HDAC2 in the regulation of MDM2 acetylation at previously uncharacterized lysines. Upon inactivation of HDAC2, this acetylation creates a structural signal in the lysine-rich domain of MDM2 to prevent the recognition and degradation of its downstream substrate, MCL-1 ubiquitin ligase E3 (MULE). This mechanism further reveals a therapeutic connection between the MULE ubiquitin ligase function and tumor suppression. Specifically, we show that HDAC inhibitor treatment promotes the accumulation of MULE, which diminishes the t(X; 18) translocation-associated synovial sarcomagenesis by directly targeting the fusion product SS18-SSX for degradation. These results uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response. : Biological Sciences; Molecular Biology; Cancer Subject Areas: Biological Sciences, Molecular Biology, Cance