Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis.

Microtubule-severing enzymes (MTSEs) play important roles in mitosis and meiosis of the primitive organisms. However, no studies have assessed their roles in mammalian meiosis of females, whose abnormality accounts for over 80% of the cases of gamete-originated human reproductive disease. In the current study, we reported that katanin-like 2 (KL2) was the only MTSE concentrating at chromosomes. Furthermore, the knockdown of KL2 significantly reduced chromosome-based increase in the microtubule (MT) polymer, increased aberrant kinetochore-MT (K-MT) attachment, delayed meiosis, and severely affected normal fertility. Importantly, we demonstrated that the inhibition of aurora B, a key kinase for correcting aberrant K-MT attachment, eliminated KL2 from chromosomes completely. KL2 also interacted with phosphorylated eukaryotic elongation factor-2 kinase; they competed for chromosome binding. We also observed that the phosphorylated KL2 was localized at spindle poles, and that KL2 phosphorylation was regulated by extracellular signal-regulated kinase 1/2. In summary, our study reveals a novel function of MTSEs in mammalian female meiosis and demonstrates that multiple kinases coordinate to regulate the levels of KL2 at chromosomes.

CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

Dendrobium huoshanense Polysaccharide Improves High-Fat Diet Induced Liver Injury by Regulating the Gut-Liver Axis.

Dendrobium huoshanense is an important Traditional Chinese medicine that thickens the stomach and intestines. Its active ingredient Dendrobium huoshanense polysaccharide (DHP), was revealed to relieve the symptoms of liver injury. However, its mechanism of action remains poorly understood. This study aimed to investigate the mechanism of DHP in protecting the liver. The effects of DHP on lipid levels, liver function, and intestinal barrier function were investigated in mice with high-fat diet-induced liver damage. Changes in the gut flora and their metabolites were analyzed using 16S rRNA sequencing and metabolomics. The results showed that DHP reduced lipid levels, liver injury, and intestinal permeability. DHP altered the intestinal flora structure and increased the relative abundance of Bifidobacterium animalis and Clostridium disporicum. Furthermore, fecal metabolomics revealed that DHP altered fecal metabolites and significantly increased levels of gut-derived metabolites, spermidine, and indole, which have been reported to inhibit liver injury and improve lipid metabolism and the intestinal barrier. Correlation analysis showed that spermidine and indole levels were significantly negatively correlated with liver injury-related parameters and positively correlated with the intestinal species B. animalis enriched by DHP. Overall, this study confirmed that DHP prevented liver injury by regulating intestinal microbiota dysbiosis and fecal metabolites.

ZP3 and AIPL1 participate in GVBD of mouse oocytes by affecting the nuclear membrane localization and maturation of farnesylated prelamin A.

The low maturation rate of oocytes is an important reason for female infertility and failure of assisted pregnancy. The germinal vesicle breakdown (GVBD) is a landmark event of oocyte maturation. In our previous studies, we found that zona pellucida 3 (ZP3) was strongly concentrated in the nuclear region of germinal vesicle (GV) oocytes and interacted with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) and lamin A to promote GVBD. In the current study, we found that lamin A is mainly concentrated in the nuclear membrane. When ZP3 is knocked down, lamin A will be partially transferred to the nucleus of oocytes. The prelamin A is increased in both the nuclear membrane and nucleus, while phosphorylated lamin A (p-lamin A) is significantly reduced. AIPL1 was also proved to accumulate in the GV region of oocytes, and ZP3 deletion can significantly inhibit the aggregation of AIPL1 in the nuclear region. Similar to ZP3 knockdown, the absence of AIPL1 resulted in a decrease in the occurrence of GVBD, an increase in the amount of prelamin A, and a significant decrease in p-lamin A in oocytes developed in vitro. Finally, we propose the hypothesis that ZP3 can stabilize farnesylated prelamin A on the nuclear membrane of AIPL1, and promote its further processing into mature lamin A, therefore promoting the occurrence of GVBD. This study may be an important supplement for the mechanism of oocyte meiotic resumption and provide new diagnostic targets and treatment clues for infertility patients with oocyte maturation disorder.

Microtubule-severing protein Fidgetin-like 1 promotes spindle organization during meiosis of mouse oocytes.

Microtubule-severing proteins (MTSPs) play important roles in mitosis and interphase. However, to the best of our knowledge, no previous studies have evaluated the role of MTSPs in female meiosis in mammals. It was found that FIGNL1, a member of MTSPs, was predominantly expressed in mouse oocytes and distributed at the spindle poles during meiosis in the present study. FIGNL1 was co-localized and interacted with γ-tubulin, an important component of the microtubule tissue centre (MTOC). Fignl1 knockdown by specific small interfering RNA caused spindle defects characterized by an abnormal length:width ratio and decreased microtubule density, which consequently led to aberrant chromosome arrangement, oocyte maturation and fertilization obstacles. In conclusion, the present results suggested that FIGNL1 may be an essential factor in oocyte maturation by influencing the meiosis process via the formation of spindles.

Fidgetin knockdown and knockout influences female reproduction distinctly in mice.

Microtubule-severing proteins (MTSPs), are a family of proteins which use adenosine triphosphate to sever microtubules. MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes. One member of this family, fidgetin ( FIGN), is also involved in male fertility; however, no studies have explored its roles in female fertility. In this study, we found mouse fidgetin is rich within oocyte zona pellucida (ZP) and is the only MTSP member to do so. Fidgetin also appears to interact with all three ZP proteins. These findings prompted us to propose that fidgetin might prevent polyspermy. Results from in vitro maturation oocytes analysis showed that fidgetin knockdown did cause polyspermy. We then deleted all three fidgetin isoforms with CRISPR/Cas9 technologies; however, female mice remained healthy and with normal fertility. Of all mouse MTSPs, only the mRNA level of fidgetin-like 1 ( FIGNL1) significantly increased. Therefore, we assert that fidgetin-like 1 compensates fidgetin's roles in fidgetin knockout female mice.

Spastin interacts with CRMP5 to promote spindle organization in mouse oocytes by severing microtubules.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

Inhibiting bridge integrator 2 phosphorylation leads to improved oocyte quality, ovarian health and fertility in aging and after chemotherapy in mice.

Female ovaries degenerate about 20 years earlier than testes leading to reduced primordial follicle reserve and a reduction in oocyte quality. Here we found that bridge integrator 2 (BIN2) is enriched in mouse ovaries and oocytes and that global knockout of this protein improves both female fertility and oocyte quality. Quantitative ovarian proteomics and phosphoproteomics showed that Bin2 knockout led to a decrease in phosphorylated ribosomal protein S6 (p-RPS6), a component of the mammalian target of rapamycin pathway and greatly increased nicotinamide nucleotide transhydrogenase (NNT), the free-radical detoxifier. Mechanistically, we find that phosphorylation of BIN2 at Thr423 and Ser424 leads to its translocation from the membrane to the cytoplasm, subsequent phosphorylation of RPS6 and inhibition of Nnt translation. We synthesized a BIN2-penetrating peptide (BPP) designed to inhibit BIN2 phosphorylation and found that a 3-week BPP treatment improved primordial follicle reserve and oocyte quality in aging and after chemotherapy-induced premature ovarian failure without discernible side effects.

Microtubule­severing protein Katanin p60 ATPase­containing subunit A­like 1 is involved in pole­based spindle organization during mouse oocyte meiosis.

Microtubule­severing proteins (MTSPs) are a group of microtubule­associated proteins essential for multiple microtubule­related processes, including mitosis and meiosis. Katanin p60 ATPase­containing subunit A­like 1 (p60 katanin­like 1) is an MTSP that maintains the density of spindle microtubules at the poles in mitotic cells; however, to date, there have been no studies about its role in female meiosis. Using in vitro­matured (IVM) oocytes as a model, it was first revealed that p60 katanin­like 1 was predominant in the ovaries and oocytes, indicating its essential roles in oocyte meiosis. It was also revealed that p60 katanin­like 1 was concentrated at the spindle poles and co­localized and interacted with γ­tubulin, indicating that it may be involved in pole organization. Next, specific siRNA was used to deplete p60 katanin­like 1; the spindle organization was severely disrupted and characterized by an abnormal width:length ratio, multipolarity and extra aster microtubules out of the main spindles. Finally, it was determined that p60 katanin­like 1 knockdown retarded oocyte meiosis, reduced fertilization, and caused abnormal mitochondrial distribution. Collectively, these results indicated that p60 katanin­like 1 is essential for oocyte meiosis by ensuring the integrity of the spindle poles.

Physicochemical characteristics and functionality of tree peony (Paeonia suffruticosa Andr.) seed protein.

The physicochemical and functional properties of tree peony seed protein were investigated. Tree peony seed protein with a favourable amino acid profile was composed of a 60kDa protein with two subunits of 38 and 23kDa. The isoelectric points of the two subunits were 3.6 and 9.0. Moreover, acid-Schiff staining indicated both of them were glycoproteins. Diagonal and 2-D electrophoresis data indicated the 38kDa subunit included three types, which two types had inter-disulphide bonds and one type had no-disulphide bonds. So did the 23kDa subunit. Circular dichroism spectra indicated the tree peony seed protein had predominantly a ß-sheet structure. Differential scanning calorimetry analysis indicated the denaturation temperatures of the tree peony seed protein at pH 5.0, 7.0 and 9.0 were 92.0, 97.1 and 95.2°C, respectively. Tree peony seed protein could be a food ingredient in the food industry due to its desirable physicochemical and functional properties.

Pnma5 is essential to the progression of meiosis in mouse oocytes through a chain of phosphorylation.

PNMA (paraneoplastic antigen MA) family includes Pnma1-6. Although other members have been found to be involved in paraneoplastic neurological disorders, death receptor-dependent apoptosis, and tumorigenesis, Pnma5 was thought to be a female fertility factor, as indicated by one genome-wide study. But until now there have not been any further functional studies about Pnma5 in female meiosis. Our preliminary study indicated that Pnma5 might play important roles in meiosis. To further address this, Pnma5 was knocked down in in-vitro maturated (IVM) mouse oocytes, which are common models for mammalian female meiosis, by specific siRNA, and results showed that the loss of Pnma5 significantly delayed the progression of meiosis I and increased chromosome segregation errors during anaphase I. In in-vitro fertilization (IVF), Pnma5 knockdown caused significantly lower fertilization. To assess how it affects meiosis, Pnma5 knockdown was found to significantly decrease the stability of spindle microtubules and altered F-actin organization within actin cap regions, cause significantly abnormal mitochondria aggregation and lower ATP concentration. Next we have found that phosphorylation at Thr533 re-located Pnma5 strongly to spindles & cortex and was required for the phosphorylation of Akt and Gsk3ß, while Src and Erk1/2 phosphorylation was required for the phosphorylation of Pnma5, indicating that phosphorylated Pnma5 is the active form and subsequently activates Akt and Gsk3ß. Collectively this study suggests that Pnma5 is important for meiosis and is the pivot of Src→Erk1/2→Pnma5→Akt→Gsk3ß pathway.

ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis.

ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD.

Crystal structure of 4-methyl-2-oxo-2H-chromen-7-yl ferrocene-carboxyl-ate.

The title mol-ecule, [Fe(C5H5)(C16H11O4)], consists of a ferrocenyl moiety and a 4-methyl-coumarin group linked through an ester unit to one of the cyclo-penta-dienyl (Cp) rings. The two Cp rings are virually parallel, with an angle between the two least-squares planes of 0.74 (16)°. The distances between the Fe(II) atom and the centroids of the two Cp rings are 1.639 (2) and 1.652 (2) Å. The conformation of the ferrocenyl moiety is slightly away from eclipsed. The dihedral angle between the coumarin ring system and the ferrocenyl ester moiety is 69.17 (19)°. π-π stacking inter-actions involving the benzene rings of neighbouring coumarin moieties, with centroid-centroid distances of 3.739 (2) Å, consolidate the crystal packing.