Kohkichi Uehara

Dynamics of Messenger RNAs Encoding Inhibin/Activin Subunits and Follistatin in the Ovary During the Rat Estrous Cycle

Abstract Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin α subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin βA subunit mRNA. On the other hand, inhibin/activin βB subunit mRNA showed a different changing pattern. Levels of βB subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, α subunit mRNA levels were considerably higher than βA and βB subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian βA and βB subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of β subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.

Characterization of Rat Follistatin-Related Gene: Effects of Estrous Cycle Stage and Pregnancy on Its Messenger RNA Expression in Rat Reproductive Tissues

Abstract Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.

Expression of Type II and Type XI Collagens in Canine Mammary Mixed Tumors and Demonstration of Collagen Production by Tumor Cells in Collagen Gel Culture

The development of cartilaginous collagen types, II and XI, in canine mammary mixed tumors was studied biochemically and immunohlstochemically. In mixed tumor, an alcian blue‐positive myxomatous region appeared in the stroma, where round‐shaped proliferating myoepithelial cells were scattered. Type II collagen was distributed in metaplastic cartilage matrix, while type XI was located only in the pericellular region, where proliferating cells were positively stained with anti‐actin and anti‐keratin antibodies. The accumulation of collagen types II and XI in the tumor mass was confirmed by sodium do decyl sulfate‐polyacrylamide gel electrophoresis followed by immunoblotting of the extract of the lesion using type‐specific antibodies to collagen types II and XI. Tumor cells isolated from metaplastic tumor mass expressed both collagen types II and XI and myoepithelial types of cytoskeleton in gel culture, in which an alcian blue‐positive substance became detectable in the pericellular region on day 3 and type II and type XI collagens on day 5. This may be a useful model for studying chondrocyte‐type gene expression during tumorigenesis .

Simultaneous Expression of Type IX Collagen and an Inhibin‐related Antigen in Proliferative Myoepithelial Cells with Pleomorphic Adenoma of Canine Mammary Glands

To identify an alcian blue‐positive component expressed in the early stage of mammary mixed tumor and to pursue the possible involvement of a tumorigenic inducing factor, monoclonal antibodies to type IX collagen were generated and used to investigate the immnnohistochemical kinetics of type IX collagen expression by the myoepithelial cell‐derived chondrocyte‐Uke cells during the development of chondrometaplasia. We also examined the expression of inhibin‐related antigen using antibodies to a synthetic peptide spanning amino acids 1–30 of the inhibin α chain. At the earliest stage of chondrometaplasia, where myoepithelial cells began to proliferate inside the basement membrane, the cells expressed type IX collagen together with an inhibin‐related antigen which was immunoreactive with the anti‐inhibin peptide antibodies. The expression of the inhibin‐related antigen was also demonstrated in normal embryonic chondrocytes and myoblasts, but was much less strong in mature chondrocytes and myotubes, strongly suggesting that the inhibin‐related antigen is involved in the development of chondrocytes and myoblasts from undifferentiated mesenchymal cells as well as proliferating myoepithelial cells as a chondro‐progenitor cell in the mammary mixed tumor. The pathophysiological significance of type IX collagen expression as a possible cell marker of the progenitor cell in myoepithelial cell‐related chondrometaplasia is also discussed.

Localization of Type IV Collagen in the Myotome Cells during the Somite Differentiation in the Chick Embryo

Type IV collagen is a major structural component of basement membranes which play various roles upon their adjacent cells. In order to better understand roles of type IV collagen during the somite differentiation, we produced anti‐rat type IV collagen polyclonal antibodies and demonstrated spacial and temporal distribution of type IV collagen in somites of the chick embryo by immunohistochemical procedure. Type IV collagen was detected in the basal surface and the cytoplasm of epithelial dermatome cells at early stage of the somite differentiation, and then detected in myotome cells overlying apical surface of dermatomes, but not in migratory mesenchymal dermatome cells. With the appearance of type IV collagen‐expressing myotome cells, epithelial dermatome cells showed the decrease in immunoreaction with anti‐type IV collagen antibodies, the disappearance of their basal‐apical polarity and their epithelial shape. From these results, it was suggested that type IV collagen is an early marker for myotome cells, and that type IV collagen and/or other factors co‐expressed by myotome cells might provide an accelerative signal for epithelial/mesenchymal conversion of dermatome cells.

Contribution of endogenous inhibin to the decline of the secondary surge of follicle-stimulating hormone in the rat

The involvement of inhibin in the decline of the secondary surge of follicle-stimulating hormone (FSH) was investigated in the rat. After ovariectomy or treatment with inhibin antiserum conducted at 2300 hours during pro-oestrus, plasma concentrations of FSH were maintained at high levels compared with control rats. However, plasma FSH started to decline at 0500 hours during oestrus in both the groups. The same treatments conducted during metoestrus markedly increased plasma FSH after 24 h (twofold compared with the treatments during pro-oestrus), suggesting that the treatments sufficiently depleted circulating inhibin. To examine whether the decline of plasma FSH occurred through a transcriptional mechanism or through a translational mechanism, FSH-beta mRNA expression and the pituitary concentration of FSH were measured. Neither ovariectomy nor inhibin immunization conducted during the night of pro-oestrus, affected the pituitary concentration of FSH after 24 h, whereas a noticeable increase was observed after the treatments conducted during metoestrus. In both stages, both ovariectomy and inhibin immunization significantly increased FSH-beta mRNA expression compared with control rats. In contrast with the pituitary concentration of FSH, the effect of inhibin immunization on FSH-beta mRNA expression was not different between the stages. The present data demonstrate the involvement of inhibin in the decline of the secondary surge of FSH, and suggest that a factor or factors other than inhibin may also be responsible for the fall in FSH. Changes in the pituitary concentration of FSH and FSH-beta mRNA expression suggest that post-transcriptional mechanisms may be involved in the suppression of FSH secretion during oestrus.

The Preparation of Leather Tanned with Saved-chrome Tanning Agent and Chrome Liquor Recycling

北米産塩蔵成牛皮を裸皮の状態で予備分割し,裸皮重量に対し3%のクロム鞣剤(Cr2O3:25%)を使用してウエットブルーを調製した.このウエットブルーを分割,シェービング後,各種の鞣剤またはクロム鞣剤で再鞣を施すことにより,クロム鞣剤の使用量を大幅に削減することが可能であった.省クロム鞣しウエットブルーから調製されたナッパ革,靴甲革,靴ソフト甲革および床ベロア革の品質は良好であった.省クロム鞣しのウエットブルーをクロム再鞣する場合,再鞣排液中のクロムは添加量の56%と高いので,これを循環利用することがクロムの使用量と排出量の削減に非常に有効であることを認めた.床ウエットブルーをクロム再鞣する場合,再鞣浴にウエットブルーを16時間浸漬することにより再鞣でのクロム利用率が向上した.