The 1,860,725-base-pair genome of Thermotoga maritima MSB8 contains 1,877 predicted coding regions, 1,014 (54%) of which have functional assignments and 863 (46%) of which are of unknown function. Genome analysis reveals numerous pathways involved in degradation of sugars and plant polysaccharides, and 108 genes that have orthologues only in the genomes of other thermophilic Eubacteria and Archaea. Of the Eubacteria sequenced to date, T. maritima has the highest percentage (24%) of genes that are most similar to archaeal genes. Eighty-one archaeal-like genes are clustered in 15 regions of the T. maritima genome that range in size from 4 to 20 kilobases. Conservation of gene order between T. maritima and Archaea in many of the clustered regions suggests that lateral gene transfer may have occurred between thermophilic Eubacteria and Archaea.

BackgroundWe have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.Methodology/Principal FindingsWhen induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.SignificanceThe ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.

The success of early embryonic development depends on oocyte nuclear and cytoplasmic maturation. We have investigated whether glial cell line-derived neurotrophic factor (GDNF) affects the in vitro maturation (IVM) of porcine oocytes and their subsequent ability to sustain preimplantation embryo development. GDNF and both its coreceptors, GDNF family receptor alpha-1 (GFR alpha-1) and the rearranged during transformation (RET) receptor, were expressed in oocytes and their surrounding cumulus cells derived from small and large follicles. When included in IVM medium, GDNF significantly enhanced cumulus cell expansion of both small and large cumulus-oocyte complexes and increased the percentage of small follicle-derived oocytes maturing to the metaphase II stage, although nuclear maturation of large oocytes was not significantly affected. Examination of cyclin B1 protein expression as a measure of cytoplasmic maturation revealed that in the presence of GDNF, cyclin B1 levels were significantly increased in large follicle-derived oocytes, as well as in oocytes from small follicles to a level comparable to the untreated large group. After activation, a significantly higher percentage of both small and large oocytes that were matured in the presence of GDNF developed to the blastocyst stage compared with untreated controls. Indeed, GDNF enhanced the blastocyst rate of small oocytes to levels comparable to those obtained for large oocytes matured without GDNF. The effect of GDNF was specific; this was evident because its enhancement of nuclear maturation and embryo developmental potential was blocked by an antibody against GFR alpha-1. Our study provides the first functional evidence that GDNF affects oocyte maturation and preimplantation embryo developmental competence in a follicular stage-dependent manner. This finding may provide insights for improving the formulation of IVM culture systems, especially for oocytes from small follicles.

Artificial transcription factors containing designer zinc-finger DNA-binding domains (DBDs) have been used to activate or repress expression of a growing number of endogenous genes. We have combined targeted zinc-finger DBD technology with a dimerizer-regulated gene expression system to permit the small-molecule control of endogenous gene transcription. We constructed a dimerizer-responsive transcription factor that incorporates an artificial zinc-finger DBD targeted to the promoter of the human VEGF gene. Introduction of this activator into human cells allowed expression of the chromosomal VEGF gene to be induced by a small-molecule dimerizer compound consisting of a nonimmunosuppressive rapamycin analog. We found that by directly regulating zinc-finger protein (ZFP) activity, we could circumvent difficulties encountered in the generation of cell lines stably expressing conventional unregulated activators. Dimerizer-dependent VEGF induction was rapid, tight, and dose dependent, and resulted in VEGF protein expression levels several-fold greater than those produced by the natural hypoxic response.

Studies of naturally occurring human GnRH receptor (GnRHR) mutants may provide a useful approach to dissecting the signal transduction pathways involved in mediating the effects of GnRH. We have analyzed two common mutations in the GnRHR, corresponding to amino acid substitutions Gln106Arg and Arg262Gln, for their effects on the stimulation of gonadotropin subunit and GnRHR gene expression by GnRH. Despite similar impairment of GnRH-stimulated inositol phosphate production, dose-response analyses indicated that Gln106Arg and Arg262Gln both reduced the sensitivity of the FSH beta gene promoter to a greater extent than LH beta or alpha GSU, suggesting the involvement of more than one signaling pathway. Furthermore, although the sensitivities of the LH beta and FSH beta gene promoters to GnRH were similarly affected by both mutants, alpha GSU sensitivity was decreased to a greater extent by Arg262Gln than by Gln106Arg. Similarly, GnRHR gene promoter sensitivity was significantly reduced only by Arg262Gln. To further characterize the differential downstream effects of these mutant GnRHRs, we investigated their effects on additional signal transduction pathways. The mutant receptors differentially affected GnRH-mediated activation of the ERK pathway and GnRH stimulation of cAMP response element-mediated transcription. These results indicate that measurement of inositol phosphate production alone may not be adequate for assessing mutant GnRHR function and additional signal transduction pathways may better reflect physiologically relevant effects. The differential stimulation of LH beta, FSH beta, and alpha GSU gene expression may contribute to the varied phenotypes observed among patients harboring these mutations.

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