Cryopreservation of canine ovaries by vitrification

The cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of working dogs, including guide dogs for the blind. However, no attempt has been reported on cryopreservation of the canine ovary. Thus, we evaluated a vitrification method for cryopreservation of canine ovaries and determined the potential functionality of vitrified-warmed canine ovaries by means of transplantation into non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. All ovarian tissues cryopreserved by vitrification were morphologically normal in terms of histology. Cryopreserved ovaries were transplanted into the ovarian bursa of the NOD-SCID mice, and the xenografts were recovered from 23 of 23 mice (100%) 4 weeks after the operation. The transplanted canine tissue was tightly adhered to the mouse ovary. Although antral follicle formation did not occur after grafting, proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. These results indicate that cryopreservation of the canine ovary by vitrification appears to have the potential to restore endocrine function and ovulation potential.     

Prominent expression of spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1 in LH-producing cells, LbetaT2

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells. We examined whether the expression of SCA1 gene in LbetaT2 cells is related to hormone production. We also compared the expression of SCA1 with that in various other pituitary tumor derived cell lines, and confirmed the prominent expression of SCA1 in LbetaT2 cells. The effect of gonadal factor(s) for SCA1 gene expression was examined. The expression level in female rats was low and did not change during the estrus cycle, but increased significantly after ovariectomy and did not return to the normal level under low and high doses of estrogen. In the male pituitary SCA1 gene expression increased markedly after castration and was not decreased by estrogen or testosterone. The Ontogeny of SCA1 gene expression was investigated in porcine fetal and postnatal pituitaries and revealed biphasic and sexually dimorphic expression. Transient expression of SCA1 gene was observed at fetal day 50 and 65 in males and day 40 in females, followed by a decline and increased expression before birth in both genders. Thus the expression of SCA1 gene is prominent in LH-producing cells and is not under direct control of gonadal factor(s) in both genders. In addition to the variable expression of SCA1 gene during the fetus period, the present results provide a novel aspect to the understanding of Boucher-Neuhauser syndrome (Ataxia Hypogonadism Choroidal Dystrophy).     

Cloning and characterization of pea apyrases: involvement of <Emphasis Type="Italic">PsAPY1 </Emphasis>in response to signal molecules from the pea pathogen <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis>

 Two nucleoside triphosphatase (NTPase) cDNA clones were isolated from a cDNA library of Pisum sativum L., cv. Midoriusui. The genes encoding the cDNAs were designated PsAPY1 and PsAPY2. PsAPY1 included the N-terminal amino acid sequence of an NTPase bound to pea cell wall. The phylogenic analysis indicated that PsAPY1 belongs to an NTPase subfamily responsive to environmental stimuli and that PsAPY2 belongs to a discrete subfamily, the physiological role of which is almost unknown. The adenosine triphosphatase activity of recombinant PsAPY1 was regulated by an elicitor and a suppressor from the pea pathogen Mycosphaerella pinodes. Based on these findings, we discuss the role of NTPases in response to biological stresses. Received: May 27, 2002 / Accepted: July 31, 2002     

High level expression of Prop-1 gene in gonadotropic cell lines

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.     

Study on the nitric oxide scavenging effects of ginseng and its compounds

In this study, an in vitro nitric oxide (*NO)-generating system was used to investigate the *NO-scavenging effects of methanolic extracts of white ginseng (Panax ginseng C.A. Meyer), red ginseng, and sun ginseng and several ginsenosides and phenolic compounds. Sun ginseng extract showed the strongest activity among the three ginseng extracts. None of the ginsenosides used in this experiment showed *NO-scavenging activity, but the phenolic compounds, such as p-coumaric and vanillic acids, and maltol inhibited *NO production in a concentration-dependent manner. Moreover, maltol levels markedly increased by heat processing. Therefore, the enhanced *NO-scavenging activity of ginseng by heat processing was closely related to phenolic acids and the increased content of maltol.     

Genetic analysis of shiga-toxigenic Escherichia coli isolates from cattle in a limited region

The ecology of shiga‐toxigenic Escherichia coli (STEC) is important in the animal production environment. We investigated fecal shedding of STEC in one town in Miyagi, Japan by multiplex polymerase chain reaction (PCR) targeting shiga toxin gene 1 (stx₁), gene 2 (stx₂) and malB promoter gene, and analyzed the PCR products of stx₁ or stx₂ (54 samples) by direct sequencing. Three of 46 (6.5%) beef cattle in the University Farm of Tohoku University (Kawatabi Farm) and 11 of 70 (15.7%) calves in neighboring dairy farms carried STEC. Rate of detecting genes of stx₁, stx₂ and stx₁₊₂ was 3.4% (4/116), 8.6% (10/116) and 0.9% (1/116), respectively. Serotyping indicated that STEC contaminated farms at different times or through different routes. Isolates harbored no mutation among stx₁, but six (Kawatabi Farm) and 38 (neighboring farms) base substitutions among stx₂, respectively. The diversity of substitutions of stx₂ was observed among farms or even in a farm. Phylogenic analysis revealed that STEC detected in the area were classified into three clusters by the variety of stx₂. Sequence analysis of stx₂ will be one of the tools for clarifying the source of outbreaks and the route of contamination of STEC.