Down-regulation of endogenous Wt1 expression by Sry transgene in the murine embryonic mesonephros-derived M15 cell line.

Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo.

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

Long term adrenal insufficiency induces skeletal muscle atrophy and increases the serum levels of active form myostatin in rat serum.

Skeletal muscle wasting is a common symptom in the adrenal insufficiency such as Addison's disease. Although it has been suspected that several cytokines and/or growth factors are responsible for the manifestation of the symptom, the precise mechanisms underlying the phenomenon have so far been poorly understood. Myostatin is predominantly expressed in skeletal muscles and involved in the regulation of skeletal muscle mass. Recently, several reports indicated that myostatin is secreted into the circulation and the increased levels of circulating myostatin is associated with the induction of skeletal muscle wasting in adult animals. We, therefore, hypothesized that the increased levels of circulating myostatin may account for the development of skeletal muscle wasting in adrenal insufficiency. To test the validity of this hypothesis, we compared the serum levels of myostatin in normal with those in bilaterally adrenalectomized (ADX) rats, a model of Addison's disease, by Western blot analysis. The active form of myostatin (13 kDa) was barely detectable in the sera collected either 1 month or 2 month after adrenalectomy, but present at conspicuously detectable levels in those obtained 3 month after the operation, while the total amounts of myostatin proteins (sum of the precursor and the active forms) remained constant at all the time points examined post-operatively. These results are consistent with the hypothesis that the increased serum levels of active form of myostatin protein, induced yet unknown post-translational control mechanisms may be responsible, at least in part, for the muscle wasting associated with the adrenal insufficiency syndromes.

Dynamics of betaig-h3 mRNA expression during pregnancy in the uterus and the placenta of the mouse: a possible regulatory factor for trophoblastic invasion.

Changes in the betaig-h3 gene expression levels in the uterus during different reproductive stages and those in the placenta on different days of gestation in the mouse were examined by Northern blot analysis. The levels of betaig-h3 expression rose steeply from the baseline level at proestrus to the maximum at estrus and then declined rapidly from 1st day of diestrus onward. During pregnancy, high levels of betaig-h3 mRNA were detected in the uterus on Day 4 of gestation, when the trophoblastic invasion of the implanted blastocysts was very active. In the pseudopregnant uterus, the betaig-h3 expression levels exhibited the same patterns as those in the pregnant uterus, although the levels were much lower at all stages than those in the pregnant uterus. In the placenta, the betaig-h3 expression levels gradually increased from Day 7 onward and peaked on Day 18 of gestation. In situ hybridization analysis of the pregnant uterine tissues revealed that the luminal as well as the glandular epithelium expressed betaig-h3 mRNA at high levels. In the placenta, high levels of betaig-h3 transcripts were detected in the decidual cells and the giant trophoblast cells. These findings suggest that betaig-h3 might play a role in regulating the invasiveness of trophoblast cells during implantation and placentation of hemochorial type when extensive trophoblastic invasion of the endometrium takes place.

Essential role of ZP molecules in tubal transport of embryos in mice.

Our understandings of the molecular and cellular mechanisms underlying tubal transport of embryos are poor. This study describes the essential role of the molecules on the zona pellucida (ZP) in the tubal transport of mouse embryos. The bovine and porcine embryos that were interspecifically transferred to the mouse oviduct were selectively retained in the oviduct and rarely transported to the uterus. Antiserum ZP3-9 against synthetic peptides that are specific for mouse ZP3, significantly interfered with tubal transport of the treated embryos. The treatment of mouse embryos with antiserum ZP2-20 against the synthetic peptides, deduced from the sequences that are conserved in the structure of ZP2 from mouse and human, also inhibited their tubal transport. Among various proteolytic and glycosidic enzymes, treatments with trypsin and beta-glucosidase prior to transfer to the oviduct, significantly interfered with the tubal transport of the enzyme-treated mouse embryos. We hypothesize that species-specific epitopes on the ZP may be recognized by the oviductal cilia and/or the epithelial cells of ducts for tubal transport.

Partial Characterization of Macromolecular Components in Fetal Bovine Serum Required for Development of Mouse Blastocysts Cultured in vitro: (blastocysts/culture/in vitro/fetal bovine serum/mouse).

Elucidation of the mechanisms underlying implantation of blastocysts in eutherian mammals have been severely hampered by the lack of a suitable system for culture of blastocysts in vitro. Successful culture methods for mouse peri-implantation embryos in vitro have been described by Hsu (1971, 1973, 1978, 1990), Chen and Hsu (1982) and Naruse et al. (1985), but these methods are too complex for routine experimental purposes. We attempted, therefore, to establish a standard culture method suitable for quantitative analysis of early embryogenesis in the mouse. Our system allows the development of peri-implantation blastocysts from the time of shedding of the zona to formation of the proamniotic cavity under well defined conditions. Using this system, the macromolecular components in the sera required for the development of periimplantation mouse blastocysts in vitro were partially characterized. Results indicated that substances with molecular weight (MW) of 30 × 103 to 100 × 103 in the serum are capable of inducing the early phase of the trophoblast spreading. Furthermore, serum factors above MW 100 × 103 were found to be essential for the successful differentiation and/or development of ICM and ectoplacental cones.

Possible Patterns of Differentiation in the Primitive Ectoderm of C3H/HeNBALB/cA Chimeric Blastocysts: An Inference from Quantitative Analysis of Coat-Color Patterns: (mouse chimeras/coat-color patterns/blastocysts/primitive ectoderm/cranio-caudal axis).

Coat-color patterns of 20 C3H/HeN++BALB/cA chimeras produced by using the same colony of mice in a series of experiments, were quantitatively analyzed, by means of a video-image analysis system developed by Tachi (1988, 1989). In those chimeras, proportion of the two allogeneic components, i.e., C3H/HeN and BALB/cA, was relatively well balanced between the right and the left halves of the pelts, whereas between the anterior and the posterior regions it was unbalanced in favor of BALB/cA components in the anterior region. To interpret the results, a computerized geometrical model intended to simulate the hypothetical conditions of chimeric germ layers during early embryogenesis, was constructed. From the model, it was proposed that the seemingly high selection pressure for the BALB/cA components in the anterior region of this particular group of chimeras, might have been caused because the initial steps of the determination of the cranio-caudal axis took place in the regions of the primary ectoderm where BALB/cA, rather than C3H/HeN blastomeres were predominant. Possible variations in the alleles of the genes regulating the germ layer differentiation, might conceivably be the cause for the observed tendencies.