Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.

NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos.

NKX2 genes in vertebrates encode a sub- family of homeodomain-containing transcription factors which regulate morphogenetic events and cell differentiation during embryogenesis. In mouse embryos several NKX2 genes are expressed in the ventral midline domains of the neuroectoderm, while other NKX2 genes are primarily expressed in the mesendoderm and mesendodermally derived organs, such as heart and gut. Within several patterning centers for tissue organization sonic hedgehog (Shh) is an important signal in the formation of ventral midline structures in vertebrate embryos. Here, we investigated the role of Shh in the embryonic expression of six different but closely related NKX2 genes in Shh null mutant mice. We found that expression of NKX2.1, NKX2.2, and NKX2.9 in neural domains requires Shh signaling, whereas NKX2.3, NKX2.5 and NKX2.6 expression in endoderm and mesoderm is independent of Shh.

Formation of Rathke's pouch requires dual induction from the diencephalon.

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.

The chromosomal normality of unfertilized oocytes from patients with polycystic ovarian syndrome.

The present study was designed to compare the cycle characteristics of in-vitro fertilization (IVF) and the chromosomal normality of oocytes in patients with polycystic ovarian syndrome (PCOS) with those of patients with tubal factor infertility. In all, 28 cycles of 24 PCOS patients and 55 cycles of 31 patients with tubal factor infertility (control) were investigated. Although a significantly greater number of oocytes were retrieved from PCOS patients (mean +/- SD: 15.6 +/- 6.4 versus 9.0 +/- 4.0, PCOS versus control group, P < 0.05), the percentage of fertilized oocytes was significantly lower in the PCOS group compared with controls (40.1 versus 73.8%, P < 0.01). The pregnancy rate per embryo transfer did not differ between the two groups. Cytogenetic analysis was performed on 74 oocytes from PCOS patients and 73 oocytes from control patients. In the PCOS group, 10 of the 74 oocytes (13.5%) demonstrated aneuploidy, four (5.4%) oocytes were diploid and six (8.1%) oocytes were metaphase II with a prematurely condensed sperm chromosome (PCC). In the tubal infertility group, nine of the 73 (12.3%) oocytes showed aneuploidy, four (5.5%) oocytes were diploid and five (6.8%) oocytes were found to have PCC. There was no significant difference in the aneuploidy, diploidy and PCC rates between the two groups. These results suggest that the reduced fertilization observed in PCOS is not attributable to chromosomal aberrations or immaturity of oocytes recruited from patients with PCOS.

Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus.

We have identified a region with characteristics of a paternal-specific methylation imprint at the human H19 locus. This region, extending from -2.0 kb upstream to the start of transcription, is heavily methylated in sperm and on the paternal allele in somatic cells. This methylation was preserved during pre-implantation. Structural analysis revealed the presence of CpG islands and a large direct repeat with a 400 bp sequence reiterated several times, but no significant sequence homology to the corresponding region of the mouse H19 gene. These findings could suggest a role for secondary DNA structure in genomic imprinting across the species, and they also present a puzzling aspect of the evolution of the H19 regulatory region in human and mouse.

Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa.

The involvement of protein kinases in platelet activating factor (PAF)-induced acrosome reaction of human spermatozoa was investigated using specific inhibitors of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). PAF (10(-9)-10(-11) M) treatment of spermatozoa enhanced the acrosome reaction in a dose-dependent manner (32 +/- 4% at 10(-9) M, 28 +/- 4% at 10(-10) M and 24 +/- 3% at 10(-11) M respectively). When spermatozoa were preincubated with PKA, PKC and PTK inhibitor (KT5720, calphostin C and genistein) for 15 min prior to addition of PAF, there was a significantly reduced acrosome reaction induced by PAF, but complete inhibition was not observed. On the other hand, combined use of three inhibitors completely inhibited PAF-induced acrosome reaction to levels of non-treated samples. These results suggest that the induction of the acrosome reaction by PAF treatment may involve the activation of PKA, PKC and PTK signalling pathways, and that interaction between these pathways may regulate complex mechanisms of PAF-induced acrosome reaction.

Analysis of the p53 gene in human choriocarcinoma cell lines.

In the present study, we analysed human choriocarcinoma cell lines for abnormalities in the tumour-suppressor gene p53 by Southern blotting, Northern blotting, non-radioisotopic single-stranded conformational polymorphism (SSCP) and complementary DNA sequencing. In all cell lines (Bewo, GCH-1, GCH-2, SCH, JAR, JEG-3, NUC-1 and HCCM-5), no p53 gene abnormality was detected by using Southern blotting. p53 mRNA of the expected size was detected in all cell lines tested by Northern blotting. SSCP analysis revealed abnormalities of p53 cDNA in the SCH cell line. Sequencing analysis of the entire coding region of the p53 gene revealed that both alleles were expressed in the JEG-3 cell line, and one of the alleles contained a point mutation (G to T) in codon 167 (Gln to His). In the NUC-1 cell line both alleles were point mutated. One allele had a point mutation (A to T) that resulted in a codon 17 change (Glu to Asp), and another had a point mutation (A to T) that caused a codon 24 change (Lys to Asn). In the SCH cell line, AGG was inserted between codon 249 and 250; this insertion resulted in an abnormal structure of the p53 protein. In three out of eight human choriocarcinoma cell lines, a p53 gene abnormality was detected. Therefore our data demonstrate that p53 gene abnormalities are associated with choriocarcinoma cell lines.