Kaoru Kohmoto

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 × 10–4 M. The flagellum was acutely bent in the opposite direction to the curve of the hook‐shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 × 10–4 M and increased abruptly at 1 × 10–3 M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8‐bromo‐cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W‐13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed. Mol. Reprod. Dev. 53:77–83, 1999.

Biosynthetic pathways of testosterone and estradiol-17β in slices of the embryonic ovary and testis of the chicken (Gallus domesticus)

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.

EFFECTS OF 1α,25‐DIHYDROXYCHOLECALCIFEROL AND CORTISOL ON THE GROWTH AND DIFFERENTIATION OF PRIMARY CULTURES OF MOUSE MAMMARY EPITHELIAL CELLS IN COLLAGEN GEL

We examined the effects of 1α,25‐dihydroxycholecalciferol (1,25‐DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10−11–10−9m) of 1,25‐DHCC stimulated growth of the cells in a collagen gel matrix culture in serum‐free DMEM+Hams F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8‐fold the control after 6d in culture. In contrast, supraphysiological concentrations (10−8–10−7m) of 1,25‐DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose‐dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25‐DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25‐DHCC also affected casein synthesis by cells cultured in a serum‐free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10−11–10−9m) and inhibiting it above 10−8m. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25‐DHCC. These results suggest a physiological role for 1,25‐DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25‐DHCC does not substitute for glucocorticoids in the differentiation of the cells.

Developmental changes of steroidogenic enzyme activities in the embryonic gonads of the chicken: the sexual difference.

Steroidogenic enzyme activities in the left ovary and the testes of 9- to 15-day-old chicken embryos were measured, and development of the activities was compared between sexes. Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary and in the testis was comparable, and did not change throughout the period examined. Activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the ovary was similar to or higher than that in the testis, depending on substrates employed. In both gonads, 17 beta-HSD activity did not change or tended to decrease from 9 to 15 days of development. On the other hand, activities of 17 alpha-hydroxylase, C-17--C-20 lyase in the ovary were three to eight times those in the testis, and aromatase activity in the ovary was definitely higher than that in the testis at all stages examined. The activities of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase significantly increased from 9 to 11 days only in the ovary. From 13 to 15 days, the activities of 17 alpha-hydroxylase and C-17--C-20 lyase markedly increased only in the testis. These results suggest that, in the gonads of developing chicken embryos, there are sexual differences in the regulation of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase activities.

Immunochemical Demonstration of αs1- and β-Casein in Mouse Mammary Glands at Early Stages of Pregnancy

We generated monoclonal antibodies (MAbs) against mouse αs1- and β-casein and used them to survey casein immunochemically in mammary glands of mice at pericoitous and pregnant stages. Two MAb-producing hybridoma cells, designated MCα1 cell and MCβ1 cell, were established. Each antibody, when used in Western blotting, recognized specifically mouse αs1- and β-casein among a wide spectrum of proteins of both a lactating mammary homogenate and mouse skim milk. Immunohistochemistry revealed αs1- and β-casein in sections of lactating mammary glands. Staining was found in substances in the lumen and cytoplasm of duct and alveolar cells, particularly in rough endoplasmic reticulum and the Golgi apparatus. Mammary glands at Days 2, 4, 6, 8, and 14 of pregnancy showed positive staining specific to both αs1- and β-casein in the lumen and cytoplasm of duct cells, whereas the glands at estrus and Day 0 of pregnancy were positive mainly for αs1-casein. Semiquantitative Western blotting analysis of both casein components in epithelial cell fractions from glands during pregnancy confirmed that intra-epithelial αs1- and β-casein changed during three phases, elevated from trace levels to detectable levels during initial stages of pregnancy (Days 0, 2, and 4), declined to lower levels during mid-pregnancy (Days 6 and 8), and then rose to high levels during late pregnancy (Day 14).

Effects of cyclic AMP and protein synthesis inhibitors on in vitro progesterone synthesis by the granulosa cells of the quail (Coturnix coturnix japonica)

Abstract 1. 1. During 3 hr of in vitro incubation, the production of progesterone by granulosa cells from the largest preovulatory follicle (F1) of the Japanese quail was stimulated by 1 mM dibutyryl cyclic AMP. 2. 2. On the other hand, the progesterone production of the granulosa cells from the second largest and the third largest follicles (F2 and F3) were autonomous, since these cells produced significant amounts of progesterone in the incubation without dibutyryl cyclic AMP, and addition of dibutyryl cyclic AMP did not further stimulate the progesterone production. 3. 3. Cycloheximide or puromycin inhibited not only the progesterone production of the granulosa cells of F1, which was due to the action of dibutyryl cyclic AMP, but also the autonomous progesterone production of the granulosa cells of F2. 4. 4. These results suggest that in the quail ovary, the labile protein may play a role for the progesterone synthesis, but is not a mediator for the action of cyclic AMP.

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.

Radioreceptor assay of serum prolactin using nitrocellulose membrane-immobilized mammary prolactin receptor.

Triton-solubilized rabbit mammary prolactin (PRL) receptor was purified by concanavalin A-agarose chromatography and immobilized on a nitrocellulose membrane. Using the membrane-bound receptor and bovine serum, the serum level of PRL was determined by radioreceptor assay (RRA). The displacement curve, obtained by serial dilutions of the serum, was parallel to that obtained in the range of 0.4 and 20 ng/ml of standard bovine PRL. Serum could be included in concentrations up to 16% of the assay buffer and, thus, the detection limit of serum PRL was about 2.5 ng/ml. PRL in the serum was determined by radioimmunoassay (RIA). The ratio of the RIA and RRA estimates of PRL was 0.99 (r = +0.99, n = 55). The gel-filtration profile of serum PRL was identical to that obtained by RIA. It was concluded that membrane-bound receptor can be used for the determination of serum PRL.

Extraction of Catecholamine from Mouse Tissues by Ion-exchange Chromatography and Its Determination by HPLC

マウスの生体試料中のノルエピネフリン(NE),シピネフリン(E)およびドーパミン(DA)は,アンバーライトCG 50により,90%以上の高回収率で抽出された.抽出されたカテコールアミンは,逆相系のカラムを用いた高速液体クロマトグラフィーと電気化学的検出器を組み合わせた方法(HPLC-ECD法)により分離され,クロマトグラム上のピークの高さから,カテコールアミン量を同時に測定することができた.ピークの高さと量の間には,高い相関(r=0.99)があり,測定内および測定問変動係数は,それぞれ2.6%と3.0%以下であった.また,最低検出量は,NEが75 fmol,EおよびDAが100fmolとなり,測定感度は鋭敏であった.さまざまなマウスの生体試料において,カテコールアミンの測定を妨害する物質は,アンパーライトCG 50により除去されており,クロマトグラム上でカテコールアミンのピークを計測することができた.以上の結果から, アンパーライトCG 50とHPLCECD法を組み合わせることにより,マウス生体試料中の微量なカテコールアミンを,正確かつ同時に測定できることが明らかになった.