Kathy J. Jackson

Mouse Germline Restriction of Oct4 Expression by Germ Cell Nuclear Factor

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

Loss of Orphan Receptor Germ Cell Nuclear Factor Function Results in Ectopic Development of the Tail Bud and a Novel Posterior Truncation

ABSTRACT The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of theGCNF gene in mice. Germ line mutation of theGCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF−/− embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF−/− embryos suffer significant defects in posterior development. Unlike GCNF+/+ embryos, GCNF−/− embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF−/− embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF−/− embryos is 13 instead of 25 as in the GCNF+/+ embryos. Interestingly, the tailbud of GCNF−/− embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF−/− embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

GCNF‐dependent repression of BMP‐15 and GDF‐9 mediates gamete regulation of female fertility

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte‐specific GCNF knockout mouse model (GCNFfl/flZp3Cre+). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor‐β signaling molecules, bone morphogenetic protein 15 (BMP‐15) and growth differentiation factor 9 (GDF‐9), were up‐regulated in GCNFfl/flZp3Cre+ females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP‐15 and GDF‐9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double‐oocyte follicles, indicative of aberrant BMP‐15/GDF‐9 expression, were observed in GCNFfl/flZp3Cre+ females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP‐15 and GDF‐9, to affect female fertility.

A-Ring Reduced Metabolites of 19-nor Synthetic Progestins as Subtype Selective Agonists for ERα

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ERα and ERβ. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5α-dihydro (5α-norethisterone and 5α-Gestodene), or 3α,5α-tetrahydro or 3β,5α-tetrahydro derivat...

The intrinsic transcriptional estrogenic activity of a non-phenolic derivative of levonorgestrel is mediated via the estrogen receptor-α

Levonorgestrel (LNG), a 19-nor-testosterone derivative, is widely used in contraceptive formulations. This compound does not bind to the estrogen receptor (ER), but it shows estrogen-like effects under in vivo and in vitro conditions. The estrogenicity of LNG may be attributed to its bio-transformation into non-phenolic metabolites. In this study, the ability of A-ring reduced LNG metabolites to activate transcription via an estrogenic mechanism of action, including differences between ER alpha and ER beta subtypes, were investigated. Transactivation assays were performed in HeLa cells transfected with expression vectors for ER alpha and ER beta and an estrogen-responsive reporter gene. Cells were also transfected with expression vectors for both progesterone receptor (PR) isoforms (A or B). As expected, the tetrahydro derivatives of LNG (3 alpha,5 alpha- and 3 beta,5 alpha-LNG) showed significantly lower PR-mediated transcriptional activities through both isoforms when compared with progesterone (P(4)) and LNG. In contrast, the 3 beta,5 alpha-tetrahydro derivative resulted in a significant activation of estrogen-dependent gene transcription. This effect was selectively confined to the ER alpha, since little if any activity could be observed with the ER beta and no antagonistic activities were demonstrated. This study provides structural and molecular clues for the well documented in vitro and in vivo intrinsic estrogenicity of 19-nor-testosterone-derived progestins and ligand requirements for ER alpha recognition.

In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17β-aminoestrogens

Estrogenic activities of the two 17beta-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor alpha (ERalpha) and the estrogen receptor beta (ERbeta). Administration of prolame and butolame to castrated animals increased significantly (P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame (P < 0.01), as judged by their corresponding IC(50) (5.4 +/- 0.65 and 66.6 +/- 2.57 micro;g/animal, respectively). In contrast, estradiol significantly shortened blood clotting times (P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC(50) 8.10 +/- 0.79 and 17 +/- 64 microg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC(50) 4.14 +/- 1.57 and 17.0 +/- 1.78 microg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERalpha, especially in the case of pentolame where little, if any, activation occurred through the ERbeta. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

Infection of bovine cells of embryonic origin by amphotropic retroviral vectors

Two amphotropic-based mouse retroviral vectors carrying the neomycin-resistance gene were used to infect four bovine cell lines. Two cell lines, bovine kidney and spleen cells, were refractory to the infection while two independent bovine cells of apparent embryonic origin were infected by the amphotropic retroviral vectors at a measurable liter. Southern blot analysis reveals the presence of neomycin-resistance gene in the G418- resistant bovine cells. The results demonstrate the successful transfer of a gene to bovine cells of embryonic origin using a murine retroviral vector system.

Development of suspension cultures for the study of epithelial cell polarity

Abstract Polarity is an important characteristic of all epithelial cells which plays a role in the regulation of vectorial transport of ions, proteins, and carbohydrates. Examination of cells exposed to microgravity during spaceflight indicates that cell polarity may be affected by the loss of gravity, but these effects have not yet been investigated directly. To maintain polarized cell functions in epithelial cells grown in cell culture, special cell culture substrata are usually required. As it is difficult to adapt this culture technique to the constraints of spaceflight hardware, alternative methods to maintain polarized epithelial cells in culture would facilitate the study of cell polarity in microgravity. The principal aim of the research project described below is to develop a novel cell culture system which allows hormon‐ally responsive, secretory epithelial cells to develop and maintain polarity when grown in suspension. If successful, it will be possible to utilize this culture system with exis...