Using the immunohistochemical technique, we attempted to identify the source of secretion of steroid hormones between the mid- and late-terms of gestation in dogs by investigating steroid converting enzymes such as cholesterol side-chain cleavage enzyme (SCC), 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17 alpha-hydroxylase/C17, 20lyase (c17), and aromatase in the ovaries and placenta. Aromatase positive cells were slightly confirmed in luteal cells in the mid-term of gestation (day 40), whereas, in the late-stage (day 50 and 60), the number of aromatase positive cells had increased. However, the oestrogen precursor (c-17 positive cells), could barely be identified in the marginal regions of the corpora lutea (CL) and completely disappeared in the late-stage of gestation. The androgen precursors, convertase SCC and 3 beta-HSD, were confirmed in all regions of the CL during the mid-stage of gestation (day 40), showing particularly strong cell reactions in the marginal region of the CL. Yet, these positive reactions of SCC and 3 beta-HSD in the marginal region of the CL disappeared in the late-stage of gestation. Moreover, it was discovered that the number of SCC and 3 beta-HSD positive cells had decreased in all regions of the CL. None of the enzymes were detected in the placenta. The above results indicated that the source of oestrogen secretion in pregnant dogs is considered to be the CL, and that, compared with the mid-stage of gestation, there was an increased number of oestrogen synthesizing cells within the CL in the late-stage. However, the biosynthetic site of oestrogen precursors from the luteal cells during the late-stage of gestation is still unknown.

Sphingosine 1-phosphate (S1P) is a platelet-derived bioactive sphingolipid that evokes a variety of biological responses. To understand the role of S1P in the central nervous system, we have examined the effect of S1P on the production of glial cell line-derived neurotrophic factor (GDNF) and growth regulation of cortical astrocytes from rat embryo. Moreover, we examined the possibility that the expression of GDNF is regulated differently in cultured astrocytes from the stroke-prone spontaneously hypertensive rat (SHRSP) than in those from Wistar kyoto rats (WKY). The mRNA expression was quantitated by RT-PCR based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme-linked immunosorbent assay. S1P induced the expression of GDNF mRNA and the production of GDNF protein in a dose-dependent manner in WKY astrocytes. Moreover, S1P increased cell numbers and induced the proliferation of astrocytes. In addition, the level of mRNA expression and protein production of GDNF was significantly lower in SHRSP than WKY astrocytes following exposure to S1P. These findings revealed that S1P augments GDNF protein production and cellular growth in astrocytes. Also, our results indicate that production in SHRSP astrocytes was attenuated in response to S1P compared with that observed in WKY. We conclude that S1P specifically triggers a cascade of events that regulate the production of GDNF and cell growth in astrocytes. Our results also suggest that the reduced expression of GDNF caused by S1P is a factor in the stroke proneness of SHRSP.

ABSTRACT. Increase in circulating vascular endothelial growth factor (VEGF) is suggested as a prognostic indicator in human patients with malignant tumors. The purpose of this study was to evaluate the clinical significance of circulating VEGF in dogs with mammary gland tumors (MGT). Both plasma and serum VEGF were significantly higher in dogs with MGT when compared with those in the healthy dogs. In dogs with MGT, the plasma and serum VEGF of the malignant group increased significantly compared with those of the benign group. Additionally, there was a significant difference between the plasma and serum VEGF in the groups with postoperative metastasis and no metastasis. Circulating VEGF is expected to be clinically available for the determination of prognosis in canine MGT. KEY WORDS: canine, mammary gland tumor, vascular endothelial growth factor.

Glial cell line-derived neurotrophic factor (GDNF) plays several important roles in the survival and recovery of mature neurons during ischemia. We examined the possibility that the expression of GDNF mRNA and the release of GDNF protein are regulated differentially in cultured astrocytes from the stroke-prone spontaneously hypertensive rat (SHRSP) compared with those from Wistar Kyoto rats (WKY) during hypoxia and reoxygenation (H/R) and after exposure to glutamate and hydrogen peroxide (H(2)O(2)). The mRNA expression was quantitated by reverse transcription-polymerase chain reaction (RT-PCR) based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real-time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme-linked immunosorbent assay (ELISA). GDNF mRNA expression and GDNF protein release at normoxia were greater in SHRSP than in WKY astrocytes. During H/R, however, the mRNA expression and protein release tended to be reduced in SHRSP compared with WKY. Glutamate and H(2)O(2) induced the expression of GDNF mRNA and the release of GDNF protein in both WKY and SHRSP in a dose-dependent manner. Levels of GDNF mRNA and protein in SHRSP were significantly lower than in WKY. These findings indicate that GDNF production in SHRSP astrocytes was low in response to H/R, glutamate, and H(2)O(2), compared with that observed in WKY. We conclude that the attenuated production of GDNF in astrocytes is involved in neuronal vulnerability in SHRSP during H/R, as GDNF production, which is stimulated by glutamate and H(2)O(2), is closely related to the protective effect against H/R-mediated neurotoxicity.

ABSTRACT. To establish an accurate method for parentage testing in dogs, microsatellite DNA repeat length polymorphisms were examined. We selected twenty microsatellite markers reported previously and examined their application for parentage testing in Beagles and Labrador Retrievers. Heterozygosity (He), Polymorphism Information Content (PIC), the probabilities of Paternity Exclusion (PE) and the combined PE were calculated from allelic frequencies of the markers. All markers amplified by polymerase chain reactions were polymorphic and many markers showed high He and PIC in the both breeds. The final combined PEs in Beagles and Labrador Retrievers were 0.999994 and 0.999920, respectively. The results suggest that the twenty markers can be applied for routine parentage testing in dogs.