Xiaojuan Meng

GDNF promotes tubulogenesis of GFRα1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line–derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor α1 (GFRα1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRα1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRα1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRα1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRα1-positive and GFRα1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides

In a genomic screen we isolated the Drosophila gene hugin (hug, cytology 87C1-2) by cross-hybridisation to a human glial cell line-derived neurotrophic factor cDNA. Upon cDNA sequence analysis and in vitro expression assays, the hugin gene was found to encode a signal peptide containing proprotein that was further processed in Schneider-2 cells into peptides similar to known neuropeptides. Two of the peptides were similar to FXPRL-amides (pyrokinins) and to the ecdysis-triggering hormone, respectively. The former displayed myostimulatory activity in a bioassay on the cockroach hyperneural muscle preparation, as well as in the Drosophila heart muscle assay. Hugin is expressed during the later half of embryogenesis and during larval stages in a subgroup of neurosecretory cells of the suboesophageal ganglion. Ubiquitous ectopic hugin expression resulted in larval death predominantly at or shortly after ecdysis from second to third instar, suggesting that at least one of the posttranslational cleavage products affects molting of the larva by interfering with the regulation of ecdysis.

Transplantation of Germ Cells from Glial Cell Line-Derived Neurotrophic Factor-Overexpressing Mice to Host Testes Depleted of Endogenous Spermatogenesis by Fractionated Irradiation

Abstract With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals. Successful depletion of endogenous spermatogenesis was achieved using fractionated ionizing irradiation. A dose of 1.5 Gy followed by a dose of 12 Gy after 24 h reduced the percentage of tubule cross-sections displaying endogenous spermatogenesis to approximately 3% and 10% as evidenced by histologic evaluation of testes at 12 and 21 wk, respectively, after irradiation. At this dose, no apparent harmful side effects were noted in the animals. Upon transplantation, GDNF-overexpressing germ cells were found to be able to repopulate the irradiated testes and to form clusters of spermatogonia-like cells resembling those found in the overexpressing donor mice. The cluster cells in transplanted host testes expressed human GDNF, as had been shown previously for clusters in donor animals, and both were strongly positive for the tyrosine kinase receptor Ret. Thus, we devised an efficient method for depleting the seminiferous epithelium of host mice without appreciable adverse effects. In these host mice, GDNF-overexpressing cells reproduced the aberrant phenotype found in the donor transgenic mice.

Transgene Insertion Induced Dominant Male Sterility and Rescue of Male Fertility Using Round Spermatid Injection

Abstract Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFRα2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.

GDNF-induced seminomatous tumours in mouse--an experimental model for human seminomas?

Glial‐cell‐line‐derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR‐α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss‐ and gain‐of‐function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF‐induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.