Lyzl4, a novel mouse sperm-related protein, is involved in fertilization.

The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the 'changed' amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.

Laparoendoscopic radical prostatectomy (LRP): stepwise transition from multi-site to single-site with the aid of the transurethral port.

PURPOSE: To describe our initial experience with laparoendoscopic radical prostatectomy (LRP) and a stepwise transition towards transurethral assisted laparoendoscopic single-site RP (TU-LESS RP). PATIENTS AND METHODS: From Jan. 2007 to Dec. 2016, 195 patients underwent RP, of which 89 patients were performed by LRP (Group A), 106 by TU-LESS RP (Group B). The peri-operative data were collected and analyzed. All data referring to patient demographics, surgery, pathology, and peri-operative outcomes were recorded. The cosmetic result was investigated by the Patient Scar Assessment Questionnaire (PSAQ). Analysis of variance or Chi squared test were adopted to analyze the data. RESULTS: 195 procedures were completed successfully. The operation time (109.6 ± 31.9 vs. 151.5 ± 87.3, P = 0.025) and anastomosis time (10.1 ± 4.8 vs. 21.8 ± 9.9, P < 0.001) of Group B was significantly reduced compared with Group A. Estimated blood loss in Group B was significantly lower than that in Group A (95.9 ± 11.1 vs. 180.2 ± 99.7, P = 0.006). About perioperative complications, Group B was also less compared with Group A (1.9% vs. 7.9%, P = 0.047). As to the usage of postoperative analgesics, Group B apparently used less than that in Group A (6.6% vs. 62.9%, P < 0.001), which is consistent with the visual analogue scale (VAS) of the two groups (1.7 ± 1.3 vs. 7.8 ± 1.1, P < 0.001). Patients in Group B were significantly more satisfied with incision healing than in group A (74.9 ± 9.3 vs. 49.7 ± 5.8, P < 0.001). There was no significant difference both in BCR rate and time between Group B and Group A. In urination control, more patients in Group B did not have urinary incontinence 3 month after RP compared with Group A (81.1% vs. 67.4%, P = 0.028). CONCLUSIONS: LESS RP is proved to be feasible for the proper patients, but it is difficult to popularized due to inconvenient operation. While by means of TU-LESS, operating difficulty can be significantly decreased. TU-LESS RP will be wildly accepted by surgeons and patients because of cosmetic satisfaction and quicker recovery.

Development of a heat shock inducible and inheritable RNAi system in silkworm.

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.

Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice.

The completely embryonic stem (ES) cell-derived mice (ES mice) produced by tetraploid embryo complementation provide us with a rapid and powerful approach for functional genome analysis. However, inbred ES cell lines often fail to generate ES mice. The genome of mouse ES cells is extremely unstable during in vitro culture and passage, and the expression of the imprinted genes is most likely to be affected. Whether the ES mice retain or repair the abnormalities of the donor ES cells has still to be determined. Here we report that the inbred ES mice were efficiently produced with the inbred ES cell line (SCR012). The ES fetuses grew more slowly before day 17.5 after mating, but had an excessive growth from day 17.5 to birth. Five imprinted genes examined (H19, Igf2, Igf2r, Peg1, Peg3) were expressed abnormally in ES fetuses. Most remarkably, the expression of H19 was dramatically repressed in the ES fetuses through the embryo developmental stage, and this repression was associated with abnormal biallelic methylation of the H19 upstream region. The altered methylation pattern of H19 was further demonstrated to have arisen in the donor ES cells and persisted on in vivo differentiation to the fetal stage. These results indicate that the ES fetuses did retain the epigenetic alterations in imprinted genes from the donor ES cells.

Wnt pathway is involved in pleomorphic adenomas induced by overexpression of PLAG1 in transgenic mice.

Pleomorphic adenoma gene 1 (PLAG1) was found frequently rearranged and activated in human salivary gland pleomorphic adenomas. It encodes a developmentally regulated transcription factor. Ectopic overexpression of PLAG1 has been proposed to play a crucial role in tumorigenesis of salivary gland pleomorphic adenomas. It was reported that PLAG1 can activate the transcription of insulin-like growth factor 2 (IGF2), functioning as a protooncogene. In this report, we show that the salivary gland tumors developed in PLAG1 transgenic mice share major histopathologic features with human pleomorphic adenomas. It was found that beta-catenin, the key component of Wnt signaling pathway, was upregulated at transcriptional level in tumors developed in 3 independent transgenic mouse lines. Immunohistochemical staining revealed that expression of beta-catenin as well as c-myc, downstream of beta-catenin in Wnt signaling pathway, was highly upregulated with overexpression of PLAG1 transgene in tumor and normal transgenic salivary gland tissues. Moreover, we found that PLAG1 can activate the transcription of mouse but not human beta-catenin in the 3T3 cells cotransfected with reporter constructs. Sequence analysis shows there are 4 PLAG1 consensus binding sites in mouse beta-catenin promoter region but not in human. Our findings provide the first in vivo evidence for the oncogenic activity of PLAG1 in pleomorphic adenoma tumorigenesis, reveal a valued animal model for human salivary gland tumors and suggest that Wnt signaling pathway may also contribute to the development of pleomorphic adenomas in transgenic mice.

[Generation of dspp-LacZ transgenic mouse founders].

PURPOSE: To establish a transgenic mouse founders in which the expression of LacZ was directed by a dentin sialophosphoprotein-specific promoter. METHODS: The DSPP-specific promoter was obtained by PCR and confirmed by sequencing, and the transgenic plasmid, pTN-DPM-LacZ, was constructed by subcloning the DSPP-specific promoter and LacZ-encoding sequence into one vector. The linearized transgenic plasmid was microinjected into the male pronucleus of the zygotes, and the microinjected zygotes were implanted to recipient pseudopregnant mice. The tail DNA of 4 week-pups was tested by PCR. RESULTS: 503 embryos were implanted to 20 recipient pseudopregnant mice, 12 of the 89 pups carrying the transgene. The establishment of the dspp-LacZ transgenic mouse line is under progress. CONCLUSION: 12 founders of the dspp-LacZ transgenic mice, in which the expression profile of LacZ should be the same as that of DSPP, were obtained by microinjection successfully, and the mouse line which is being established could be used as a good tool to investigate the exact expression profile of DSPP in the future.

Disruption of palladin results in neural tube closure defects in mice.

Palladin is a newly identified actin-associated protein which was proposed to be involved in actin cytoskeleton organization and nervous system development. Here, we show that inactivation of palladin leads to embryonic lethality due to severe defects of cranial neural tube closure and herniation of liver and intestine. It was found that palladin(-/-) embryos died around E15.5 and developed cranial neural tube closure defects (NTDs) with 100% penetrance. Whole mount in situ hybridization revealed that expression of palladin in early wild type embryos (E8.5) was specifically restricted in the elevating cranial neural folds where the neural tube closure is initiated. Palladin expression closely mirrors the phenotypic defects observed in palladin(-/-) mutants. While in E 9.5 and E10.5 embryos palladin was ubiquitously expressed. In vitro study revealed that formation of stress fibers in cytoplasm, cell adherent ability to extra-cellular matrix protein fibronectin and cell migration were dramatically disturbed in palladin(-/-) murine embryonic fibroblast cells (MEFs). Our findings suggest that palladin plays important roles in actin stress fiber formation, cell adhesion and migration. We propose that palladin is required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the etiology of human NTDs.