Hideya Kuroda

Infertility due to growth arrest of ovarian follicles in Sl/Slt mice.

Sl, Sld, and Slt are mutant alleles at the steel locus. All Sl/Sld and most Sl/Slt female mice are infertile, but the cause of the infertility is different. Germ cells are absent in Sl/Sld ovaries but present in Sl/Slt ovaries. The infertility of Sl/Slt female mice was attributed to the growth arrest of ovarian follicles, and the mechanism was analyzed by producing aggregation chimeras between Sl/Slt and +/+ embryos. Sl/Slt oocytes were ovulated and fertilized in Sl/Slt----+/+ chimeras. We investigated the origin of granulosa cells in the growing follicles and that of granulosa-derived luteal cells in the chimeras by using the electrophoretic pattern of phosphoglycerate kinase-1 and the histochemical activity of beta-glucuronidase as markers. Granulosa cells of Sl/Slt genotype developed and constituted pregnant corpora lutea in Sl/Slt----+/+ chimeras. Therefore, the growth arrest of Sl/Slt ovarian follicles may not be due to an intrinsic defect in granulosa cells but may instead be due to an intrinsic defect in ovarian stromal cells. This suggests that normal stromal cells are essential for the development of ovarian follicles.

Clinicopathological study of livers from brain-dead patients treated with a combination of vasopressin and epinephrine

Studies were made on the pathological lesions and biochemical indices of the livers of 22 patients in whom normal hemodynamics was maintained for 0-48 days after brain death by administration of vasopressin and epinephrine. Thirty-one specimens of liver tissues were obtained by percutaneous biopsy or at autopsy. The degrees of central venous congestion, central fibrosis, focal fibrosis, fatty metamorphosis, piecemeal necrosis, periportal fibrosis, and intrahepatic cholangitis in livers on various days after brain death were compared with those on the day of brain death (day 0). Central venous congestion was extensive on days 0-4, significantly less on days 5-14, and then again extensive on days 15-48. Central fibrosis and focal fibrosis showed no remarkable change during the 48-day period. Fatty metamorphosis, piecemeal necrosis, and periportal fibrosis showed no significant changes until day 16, but spread extensively on days 40-48. Intrahepatic cholangitis was scarcely observed on day 0 but began to increase after day 3, and spread extensively after day 5. The level of serum glutamic pyruvic transaminase did not increase in most patients until day 15. The mean value of prothrombin activity also did not decrease until day 15. However, the mean value of serum alkaline phosphatase increased gradually after day 3, and was correlated with cholangitis. The present study showed that during prolonged hemodynamic maintenance of brain-dead patients, pathological lesions did not spread or diminished and that biochemical indices did not become worse, or improved, in the first 2 weeks, except for increases in cholangitis and the serum alkaline phosphatase level.

Augmentation of aromatase activity by FSH in ovaries of fetal and neonatal mice in organ culture

The stimulative effect of FSH on aromatase activity was investigated in ovaries of fetal (on days 17 and 18 of gestation) and neonatal mice (on days 0, 3, 6 and 9 after birth). Two to six ovaries, were cultured for 48 h in 2 ml of Medium 199 supplemented with insulin (5 micrograms/ml) and [1 alpha, 2 alpha, 6 alpha, 7 alpha, beta-3H]4-androstene-3,17-dione (0.35 microM) in the presence or absence of porcine FSH (0.5 units/ml) and the amount of [3H]oestradiol-17 beta and [3H]oestrone produced was estimated. In the presence of FSH, aromatase activity per ovary, which was found in all fetal and neonatal ovaries examined, increased with age. In the absence of FSH, however, the production of oestrogens could be demonstrated only in ovaries from 3- to 9-day old mice. FSH increased the aromatase activity by up to 10-fold. In spite of the stimulative effect of FSH on aromatase activity, FSH exerted no significant effect on DNA synthesis of the ovaries. The formation of primordial follicles could not be observed histologically in ovaries of fetal mice on day 17 of gestation, although the ovaries of 6- and 9-day old mice contained multilayered follicles. These results show that FSH stimulates the aromatase activity of the mouse ovary even before the formation of primordial follicles and that the stimulative effect of FSH on ovarian aromatase is not due to the proliferation of ovarian cells.

Urinary retention induced by estrogen injections in mice: an analytical model.

Daily subcutaneous injections of pharmacological doses of 17 beta-estradiol (E2, 0.4 micrograms./gm. body weight) resulted in significant urinary retention in the bladder of castrated mice. Although the urinary retention developed in both male and female mice, the increase in urethral resistance to urinary flow and the dilatation of posterior urethra were observed only in castrated male mice receiving E2. Histological changes common to male and female mice were cornification and stratification of urethral epithelium and fibrosis of connective tissues surrounding the urethra, suggesting that these changes may cause the urinary retention. Although the exact mechanism has not been defined, the urinary retention produced by the present method may be useful as a model of human disease.

A CASE OF NONTRAUMATIC CLOSTRIDIAL GAS GANGRENE OCCURRING IN A PATIENT WITH COLON ADENOCARCINOMA, LIVER CIRRHOSIS, AND DIABETES MELLITUS

An autopsy case of clostridial gas gangrene occurring in a 54‐year‐old man with colon adenocarcinoma, liver cirrhosis, and diabetes mellitus is reported. The patient died 4 days after the onset of symptoms with episodes of vomiting and abdominal pain. Gangrene of both hips and perineum, hemolysis, renal failure, and disseminated intravascular coagulation were the dominant clinical features. Clostridium septicum was isolated from the subcutaneous tissue fluid. Adenocarcinoma of the ascending colon with ulceration found at autopsy was supposed to be an entry of the organism. Histologically, lesions of subcutaneous tissue and muscles were characterized by the absence of inflammatory infiltrates in spite of extensive necrosis. A summary of 35 cases of gas gangrene hospitalized to the Osaka University Hospital for the past 16 years indicates that clostridial gas gangrene patients with underlying diseases such as malignant neoplasm, diabetes, liver cirrhosis or immunodeficiency have a relatively poor prognosis.

Effect of genetically defined oocyte depletion on production of androgens and oestrogens by ovaries of suckling mice.

Steroid production and histological features of ovaries were compared either among normal +/+ mice of 3-12 days of age or among 12-day old mutant mice with various degrees of oocyte depletion. Whole ovaries were cultured in the medium containing [3H]progesterone and hCG or 4-androstene-3,17-dione and FSH; amounts of [3H]androgens or oestrogens released from the ovaries were assayed. FSH-responsive aromatase activity was detectable in ovaries of +/+ mice on day 3 after birth (2.6 +/- 0.4 pmol/2 ovaries/48 h), but the activity producing androgens from progesterone, under stimulation of hCG, was not detectable even on day 6 after birth (less than 0.1 pmol/2 ovaries/48 h). The androgen-producing activity appeared on day 9 after birth (1.16 +/- 0.25 pmol/2 ovaries/48 h), when follicles with more than two layers of granulosa cells developed. The ovaries of 12-day old Sl/Slt mice contained a considerable number of follicles with a single layer of granulosa cells, but did not contain any follicles with more than two layers of granulosa cells. The ovaries of Sl/Slt mice possessed aromatase activity (3.3 +/- 0.4 pmol/2 ovaries/48 h) but, not androgen-producing activity (less than 0.1 pmol/2 ovaries/48 h). The present results suggest that development of follicles with more than two layers of granulosa cells may induce the activity producing androgens from progesterone under stimulation of LH in suckling mouse ovaries, though the FSH-responsive aromatase activity is present even in follicles with a single layer of granulosa cells.

Growth competition between W mutant and wild-type cells in mouse aggregation chimeras

The dominant spotting (W) locus of the mouse has been demonstrated to be identical with the c‐kit proto‐oncogene. The c‐kit is strongly expressed in hematopoietic organs and the brain of mice. In homozygotes and double heterozygotes of the W mutant alleles (hereafter W mutant), development of erythrocytes, mast cells, melanocytes and germ cells is deficient. The deficiency of erythrocytes, mast cells and melanocytes is attributed to a defect of precursor cells, but the cause of the germ cell deficiency is not clear. We investigated the effect of the W mutation on proliferative potential of cells composing various organs by examining aggregation chimeras between W mutant and wild‐type (+/+) embryos. Proportions of +/+ components were significantly greater in the male germ cells and hematopoietic cells. In contrast, the average proportions of +/+ components were comparable to those of W mutant components in other organs including the brain. The present result suggests that the W (c‐kit) gene plays an important role in development of the male germ cells and hematopoietic cells and that it does not promote the proliferation of major cell population in the brain, in spite of the strong expression of the W (c‐kit) gene in the brain.

Effect of serum and 12-o-tetradecanoyl-phorbol-13-acetate on fsh-stimulated conversion of 4-androstene-3,17-dione to oestrogens in cell and organ cultures of suckling mouse ovaries

The effect of serum and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the FSH-stimulated oestrogen production was studied in both cell and organ cultures. Ovaries were removed from (WB X C57BL/6)F1 mice at 9-days of age, and the conversion of 4-androstene-3,17 -dione to oestrogens was stimulated by the addition of FSH in vitro. Either 10% serum (fetal calf, mouse, rat and horse) or 0.1 microM TPA markedly inhibited the FSH-stimulated oestrogen production by dispersed and cultured ovarian cells. In contrast, neither serum nor TPA influenced the oestrogen production in the organ culture. This suggests that the presence of tissue architecture may prevent the inhibitory effect of serum and TPA on the FSH-stimulated oestrogen production.

Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras

Giant lysosomal granules of bgJ/bgJ mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bgJ/bgJ genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β‐glucuronidase activity. Tubules composed of bgJ/bgJ‐type epithelial cells alone, tubules composed of +/+‐type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bgJ/bgJ‐type or +/+‐type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bgJ/bgJ‐type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bgJ/bgJ‐type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.