Cofilin phosphorylation and actin reorganization activities of testicular protein kinase 2 and its predominant expression in testicular Sertoli cells.

We previously identified testicular protein kinase 1 (TESK1), which phosphorylates cofilin and induces actin cytoskeletal reorganization. We now report identification and characterization of another member of a TESK family, testicular protein kinase 2 (TESK2), with 48% amino acid identity with TESK1. Like TESK1, TESK2 phosphorylated cofilin specifically at Ser-3 and induced formation of actin stress fibers and focal adhesions. Both TESK1 and TESK2 are highly expressed in the testis, but in contrast to TESK1, which is predominantly expressed in testicular germ cells, TESK2 is expressed predominantly in nongerminal Sertoli cells. Thus, TESK1 and TESK2 seem to play distinct roles in spermatogenesis. In HeLa cells, TESK1 was localized mainly in the cytoplasm, whereas TESK2 was localized mainly in the nucleus, which means that TESK1 and TESK2 likely have distinct cellular functions. Because the kinase-inactive mutant of TESK2 was localized in the cytoplasm, nuclear/cytoplasmic localization of TESK2 depends on its kinase activity. A TESK2 mutant lacking the C-terminal noncatalytic region had about a 10-fold higher kinase activity in vitro and, when expressed in HeLa cells, induced punctate actin aggregates in the cytoplasm and unusual condensation and fragmentation of nuclei, followed by apoptosis. Thus, we propose that the C-terminal region plays important roles in regulating the kinase activity and cellular functions of TESK2.     

Haploid male germ cells show no susceptibility to transformation by simian virus 40 large tumour antigen in transgenic mice.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.     

Transgenic Mice Over-expressing Endothelin-1 in Testis Transactivated by a Cre/loxP System Showed Decreased Testicular Capillary Blood Flow

It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals. Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner. In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1alpha (hEF1alpha) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system. In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression. To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos. These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology. Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.     

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.     

Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.     

The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice

The quality control of sperm is critical for efficient reproduction. In germ cells, cell death involves different processes to those in somatic cells, and in many cases, the trigger to induce cell death in deficient germ cells is still unclear. It is known that the fatty acid composition of sperm is related to fertility. Composition of the fatty acid of germ cells changes dynamically during spermatogenesis, and fatty acid binding protein (FABP) may be involved in these changes. In this study, we developed transgenic mice with a testicular germ-cell-specific FABP (PERF15) transgene, whose expression was controlled by the Cre-LoxP site-specific recombination system. We also developed transgenic mice with the Cre gene under the control of the spermatocyte specific Pgk2 promoter. In double transgenic mice, following Cre-mediated recombination of the PERF15 containing transgene, PERF15 was strongly overexpressed. Its overexpression induced multinucleate symplasts to form, indicating programmed germ cell death occurred at the elongated spermatid stage. As a result, sperm harboring the transgene were significantly decreased, but the surviving sperm demonstrated higher fertility than natural sperm. Therefore, we conclude that PERF15 associate with the direction of germ cell fates and preserve the quality of sperm.     

Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

Actopaxin interacts with TESK1 to regulate cell spreading on fibronectin

The focal adhesion protein actopaxin contributes to integrin-actin associations and is involved in cell adhesion, spreading, and motility. Herein, we identify and characterize an association between actopaxin and the serine/threonine kinase testicular protein kinase 1 (TESK1), a ubiquitously expressed protein previously reported to regulate cellular spreading and focal adhesion formation via phosphorylation of cofilin. The interaction between actopaxin and TESK1 is direct and the binding sites were mapped to the carboxyl terminus of both proteins. The association between actopaxin and TESK1 is negatively regulated by adhesion to fibronectin, and a phosphomimetic actopaxin mutant that promotes cell spreading also exhibits impaired binding to TESK1. Binding of actopaxin to TESK1 inhibits TESK1 kinase activity in vitro. Expression of the carboxyl terminus of actopaxin has previously been reported to retard cell spreading. This effect was reversed following overexpression of TESK1 and was found to be dependent on an inability of actopaxin carboxyl terminus expressing cells to promote cofilin phosphorylation upon matrix adhesion and caused by retention of TESK1 by this actopaxin mutant. Thus, the association between actopaxin and TESK1, which is likely regulated by phosphorylation of actopaxin, regulates TESK1 activity and subsequent cellular spreading on fibronectin.     

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

嗜铬颗粒蛋白A基因的反义转基因小鼠模型的建立及该基因启动子的组织特异性

构建小鼠嗜铬颗粒蛋白A（Chromogranin,A,CGA)基因的反义DNA载体pGAS1C－lacZ。用电穿孔的方法将该载体转化大鼠肾上腺髓质细胞瘤细胞系PC－12,X－Gal染色后证明位于CGA基因启动子下游的报告基因lacX已经表达。用限制性内切酶除去载体的质粒骨架后,显微注射入供体昆明小鼠的受精卵中,随后将注射过DNA的受精卵移植入假母的输卵管中,完成正常的胚胎发育。用PCR的方法筛选假母产下的小鼠,得到CGA基因反义RNA基因首建鼠14只。将首建鼠分别与正常昆明鼠交配,产生后代。取首建鼠的肾上腺进行X－Gal染色,组织用于石蜡切片,根据各鼠肾上腺切片的蓝色深浅判定转入基因量的高低,筛选到两只表达量高的首建鼠,留下它们的后代。取转基因鼠的各种组织用于X－Gal染色,发现报告基因在肾上腺、胰腺的胰岛中有表达,而在肌肉、脂肪组织中无表达,说明CGA基因的启动子具有神经内分泌组织特异性。