Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells.

Background: We 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.

Porcine DAZL messenger RNA: its expression and regulation during oocyte maturation.

Deleted in Azoospermia-Like (DAZL) is known to play an important role during both spermatogenesis and oogenesis, as mutations in this gene may result in male and female sterility. In order to study the expression of DAZL in the pig, we cloned the full-length coding sequence and determined its mRNA and protein expression profile in the ovary and in oocytes undergoing in vitro maturation (IVM). Immunohistochemisty revealed that DAZL protein localizes to oocytes of both preantral and antral follicles.

An epigenetic mechanism regulates germ cell-specific expression of the porcine Deleted in Azoospermia-Like (DAZL) gene.

The Deleted in Azoospermia-Like (DAZL) gene is specifically expressed in fetal and adult gonads. While DAZL is known to play a role during gametogenesis, the mechanisms governing its germ cell-specific expression remain unclear. We identified the 5' untranslated region (UTR) of the porcine DAZL gene and cloned and characterized 2 kilobase pairs of its TATA-less 5' flanking region, identifying CpG-rich regions within the proximal promoter.

Glial cell line-derived neurotrophic factor: an intraovarian factor that enhances oocyte developmental competence in vitro.

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.

Pharmacological regulation of the insulin receptor signaling pathway mimics insulin action in cells transduced with viral vectors.

Diabetes mellitus derives from either insulin deficiency (type I) or resistance (type II). Homozygous mutations in the insulin receptor (IR) gene cause the rare leprechaunism and Rabson-Mendenhall syndromes, severe forms of hyperinsulinemic insulin resistance for which no therapy is currently available. Systems have been developed that allow protein-protein interactions to be brought under the control of small-molecule dimerizer drugs.

Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function.

In the present study, we performed functional analyses of four mutations in the human GnRH receptor (GnRHR) gene, identified in patients with idiopathic hypogonadotropic hypogonadism. These mutations result in amino acid substitutions in the extracellular N-terminal domain (Thr32Ile), second extracellular loop (Cys200Tyr), third intracellular loop (Leu266Arg) and sixth transmembrane helix (Cys279Tyr). Immunocytochemical analysis of cells transfected with HA-tagged GnRHR constructs revealed that all four mutant receptors were present on the cell surface.

Two common naturally occurring mutations in the human gonadotropin-releasing hormone (GnRH) receptor have differential effects on gonadotropin gene expression and on GnRH-mediated signal transduction.

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.

Regulation of endogenous gene expression with a small-molecule dimerizer.

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.