Changes in the level of epidermal G2-chalone and mitotic activity in the rat vaginal epithelium]

The content of tissue-specific inhibitor of mitosis in epidermal epithelium (G2-chalone) was estimated by a single radial immunodiffusion test in the rat vagina during various stages of the estrous cycle. The level of chalone was found to correlate with the mitotic index (MI) of vaginal epithelium. The lowest level of G2-chalone is detected in proestrus and the highest one in estrus. The level of G2-chalone in vaginal epithelium was shown to be significantly decreased in aging rats (14--16 month-old) with regular cycles as compared to that in young normal cycle rats (3--4 month-old). The single injection of estradiol benzoate (1 microgram/100 g) into ovariectomized rats led to an increase in the MI following 18 hours. The increased MI is preceeded by a substantial drop of the G2-chalone level 12 hours after estrogen injection.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

Immunomorphologic analysis of G2-chalone localization in epidermal-type tissues

Localization of the epidermal G2-chalone in tissues has been established by means of indirect method of Coons using a monospecific immune serum. It has been found in dermal epithelia of the back, external ear, tongue, esophagus, forestomach, vagina, hairy follicles, but it has not been found in the sebaceous glands and derma. These findings are fully in agreement with the results obtained by the method of double diffusion after Ouchterlony. Tissue specificity of G2-chalone is proved by the fact that at places where epithelia differing histogenetically join with each other, it is found only in the epithelia of the epidermal type. Within the epithelial layer G2-chalone is mainly localized in the spinous and partly in the basal cells. Possible mechanisms on regulation of the mitotic activity are discussed in connection with the data obtained.     

Effect of rat liver chalones on liver cell proliferation at different times after partial hepatectomy]

The action of rat's liver ethanol extract (72--81 per cent saturation) on cell proliferation of this organ at various periods after a partial hepatectomy has been studied. The most sensitive periods of the action of G1- and G2-chalone were, resp., the time of cell transformation, and the middle of the premitotic period of cell cycle. The action of G2-chalone used is organ-specific, since the drug decreased the mitotic activity of both hepatocytes and stromal cells. At the same time, the proliferation of ear, tongue and small bowel epithelial cells remained unchanged.     

Integration of tissue-specific inhibitors and thyroid hormones in the regulation of proliferation of gastric gland epithelial cells]

G1- and G2-chalone effects of pig's stomach mucosa extract were registered in mice stomach gland epithelium. The inhibitory activity of chalones on cell's proliferation reduced following increased level of thyroid hormones. Local (chalones) and organism (thyroid hormones) factors cooperate in control of gland epithelial cell proliferation.     

Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G1-chalone, a tissue-specific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G1-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentration-independent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G1-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase alpha, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G1-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

Purification and characterization of epidermal G2- and G1-chalones]

Highly purified epidermal G1- and G2-chalones from rat skin inhibit the entering of epidermocytes to S and M phases of cell cycle respectively. Their biological activity is characterized by tissue-specificity and not by species-specificity. Both of them are tissue-specific glycoproteins as for their antigenic properties. Molecular weight of G1-chlone is 21 000, G2-chalone--34 000, isoelectric point (pH) 5.55 and 5.85 respectively. G2-chalone is the fastest as compared to G1-chalone in 5% acrylamide gel electrophoresis, pH 8.3. When injected in rabbits, G2-chalone produced monospecific antibodies which have no cross-reactivity with G1-chalone. The amino acid composition of both chalones and immunofluorescent localization of G2-chalone in epidermal tisues are given.     

Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.     

Change in the activity of epidermal chalones during skin reaction to the effect of a carcinogen and epilation]

The biological activity of an epidermal G2-chalone extracted from the rat back skin was found to decrease 1 to 2 days after the treatment with methylnitrosourea (a carcinogen), and one day after the epilation. Results of quantitative recording and morphological analysis of the skin serving as the source of chalones, pointed to different mechanisms which underlay this effect.     

Mitotic activity of gonadotropes in the anterior pituitary of the castrated male rat

Summary The proliferation of gonadotropes in the anterior pituitary of the castrated male rat was examined immunohistochemically after colchicine treatment. The results show a more than 10-fold increase in mitotic frequency in gonadotropes 1 or 2 weeks after castration, as compared with controls. This result explains the increase in the population of immunoreactive LH cells in castrated male rats. The gonadotropes decreased significantly 1 month after castration. The mitotic activity of gonadotropes was almost completely suppressed in castrates implanted with a silastic tube filled with testosterone.     

Selection of 19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958): a potent aromatase inhibitor in vivo.

19-Mercaptoandrost-4-ene-3,17-dione (ORG 30365) has been reported to be both a competitive and irreversible inhibitor of aromatase. In comparison to the known aromatase inhibitors 4-hydroxy-androst-4-ene-3,17-dione (4OH-AD) and 1-methyl-1,4-androstadiene-3,17-dione (SH 489), ORG 30365 was found to be, respectively, about 16 and 8 times more active in vitro using human placental microsomes. Although the activity profile of ORG 30365 is very attractive, this compound was not selected for further development because it has limited pharmaceutical stability, which is probably due to its free--SH group and therefore a number of more stable dithio-derivatives of ORG 30365 have been synthesized. These derivatives are considered to be converted to ORG 30365 before they become active. The in vivo aromatase inhibiting activity of these derivatives was determined in hypophysectomized rats treated with the estrogen precursor dehydroepiandrosterone sulphate (DHEAS) using inhibition of cornification of vaginal epithelium as parameter. The 19-(ethyldithio)-derivative (ORG 30958) appeared to be the most active inhibitor in this series being twice as active as ORG 30365 and about 8 times as active as inhibitors like 4OH-AD and SH 489. Besides inhibition of cornification of vaginal epithelium ORG 30958 decreased ovarian aromatase and plasma E2 levels in DHEAS-treated hypophysectomized rats. Plasma estradiol levels were also lowered by ORG 30958 in dogs which were treated with pregnant mare serum gonadotrophin in order to induce pro-estrus. ORG 30958 displayed much less than 1/400th of the androgenic activity of testosterone propionate in immature castrated rats and appeared to be devoid of estrogenic and anti-estrogenic activity in ovariectomized mature rats. A twice daily dose of 1.5 mg ORG 30958/kg postponed ovulation in mature female rats. In conclusion: ORG 30958 is a potent aromatase inhibitor in vivo. It probably becomes active after cleavage of the -S-S- bond yielding ORG 30365 a potent irreversible aromatase inhibitor. ORG 30958 does not display other hormonal activities making it an attractive candidate for the treatment of estrogen-dependent diseases.     

Behavioral action of estrogen in male hamsters: effect of the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD)

This study examines three independent behavioral variables known to be activated by testosterone in the male hamster; namely, the tendency to approach the female, the tendency to leave the female, and the amount of sniffing directed to her. Both intact and testosterone-maintained castrated male hamsters were given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in subcutaneous, Silastic capsules. In intact males, there was an ATD dose-dependent increase in the tendency to leave the female and a decrease in the amount of olfactory investigation. The tendency of the male to approach the female was unaffected. The higher doses of ATD also abolished the ability of males to discriminate between females using vaginal odor cues. These results were confirmed in castrated males whose behavior was maintained at the intact level by testosterone implants. In addition, in both intact and castrated males, estradiol or diethylstilbestrol was able to reverse the behavioral changes induced by ATD. Our results indicate that estrogen produced by aromatization of testosterone activates behavior. We conclude that estrogen, by influencing some, but not all, components of masculine behavior, has a specific role in modulating male-female interactions.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

The effect of urine from castrated male or female guinea pigs and rats on the estrous cycle of guinea pigs and rats, respectively]

A 24-hour reduced cycle duration was observed in 5-day cyclic female rats exposed to the odor of urine from male or female castrated rats. A decrease in the duration of the period of vaginal closure, ranging from 2 to 5 days, was observed in female guinea pigs exposed to the odor of urine from male or female castrated guinea pigs. The pheromonal activity of urine in both species was concluded to be no dependent upon the gonadal function.     

Fractionation of a chalone-containing substance from Ehrlich's ascites tumor using high pressure chromatography]

The effect of various fractions of chalone--containing preparation from ascyte Ehrlich's tumour obtained by high performance liquid chromatography (HPLC) on mitotic activity and DNA synthesis in the tumour has been studied. After filtration the division of active chalone component which inhibits entering cells into M-phase and S-phase took place. The component inhibiting DNA synthesis eluated with G1-chalone.     

Modulation of hepatic glucose-6-phosphate dehydrogenase activity in male and female rats by estrogen

The effect of estradiol-17 beta on the activity of glucose-6-phosphate dehydrogenase was studied in both male and female rats to further characterize the sex differences in the activity of this enzyme. Four groups of intact and castrated rats were implanted subcutaneously with graded doses (2.4, 4.8 and 7.2 micrograms/day) of pelleted estradiol in a physiologically relevant experimental system. After fourteen days the rats were sacrificed and their livers were assayed for G6PD activities. The result indicated that: (i) the enzyme activity was 3-fold higher in normal adult female than in male rats, (ii) low doses of E2 (2.4, 4.8 and 7.2 micrograms/day) increased the activity of G6PD 6-fold in castrated males and over 2-fold in female castrates as well as intact rats (iii) E2 stimulation of G6PD activity appears to be more effective in castrated males than in female rats (IV) sex difference in the activity of G6PD disappeared after treatment with E2 in castrated rats. It is concluded that the activity of G6PD in rats is markedly enhanced by low doses of E2, which appears to be largely responsible for the sex differences in the activity of this enzyme in rats.     

A system for the study of epidermal G1-chalone in cell culture. The rat tongue epithelial cell line RTE2

A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number. Three other cell lines showed essentially no growth inhibition during chalone treatment. The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo. Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed. Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h. The inhibitory effect was dose-dependent up to 3 micrograms/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml. Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h. These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal. The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.     

Quantitative determination of the epidermal G2-chalonelike factor in rat tissues by means of radial immunodiffusion]

A high-molecular glycoprotein (epidermal G2 chalone) shown to be homogeneous by electrophoresis and immunochemistry was isolated from rat skin. It exerts a strong tissue-rather than species-specific antimitotic effect on the keratinizing epithelium. The paper is concerned with its quantitative immunodiffusion determination in some tissues by means of monospecific antiserum. A clear-cut correlation between the mitotic index and the epidermal G2 chalone level in the mucosa is shown with special reference to vaginal epithelium of rats during the estrual cycle.     

Effect of serum estradiol and leptin levels on thyroid function, food intake and body weight gain in female Wistar rats

We evaluated the interplay among estrogen, leptin and thyroid function in the regulation of body mass in female rats. Adult female rats were divided into four groups: control (C, sham-operated), ovariectomized (OVX), ovariectomized treated with estradiol benzoate (Eb) 0.7 or 14 μg/100 g bw per day, during 21 days. OVX led to an increase in body mass, food intake and food efficiency (change in body mass as function of the amount of food ingested) which were normalized by the lower Eb dose, and decreased significantly when the higher dose was given. Serum leptin levels were increased more than two-fold in all ovariectomized groups. Serum T4 levels of the Eb treated OVX were significantly lower than in the controls. Serum T3 and TSH were unaffected by OVX or by Eb treatment. Uterine type 2 iodothyronine deiodinase (D2) activity changed in parallel with serum estradiol: decreased after OVX, returned to control levels after the lower E2 treatment, and increased significantly after the high Eb dosage. The hypothalamic D2 activity was reduced around 30% in all castrated groups, treated or not with estrogen, whereas in the brown adipose tissue the enzyme was not changed. Interestingly, although estrogen-treated OVX rats had lower body weight, serum leptin was high, suggesting that estrogen increases leptin secretion. Our results show that estradiol is necessary for the hypothalamic action of leptin, since the increase in leptin levels observed in all ovariectomized rats was associated with a decrease in food intake and food efficiency only in the rats treated with estrogen.     

Indomethacin inhibits cell proliferation in the oxyntic epithelium of the rat

The aim of this investigation was to examine the action of parenteral indomethacin and oral prostaglandin E₂ on cell proliferation in the rat oxyntic mucosa. Groups of Sprague Dawley rats were treated with either 1.5 mg/kg indomethacin subcutaneously, 5 mg/kg oral prostaglandin E₂ or placebo, twice daily during 5 days. All rats were killed exactly 4 hours after mitotic arrest with vincristine, and a biopsy specimen from the oxyntic mucosa was processed for routine microscopic evaluation.Mitotic figures were distributed cluster-like along the oxyntic mucosa alternating with mitosis-free areas. The total number of mitotic figures in 8 mm of mucosa was significantly reduced by administration of indomethacin (p<0.05). In rats given indomethacin, 32.5% of the examined mucosa did not have mitotic figures, which is significantly higher than 14.3% as observed in placebo-treated rats (p<0.05). Both rats treated with indomethacin and with prostaglandin E₂ had fewer microscopic fields containing 5–6 mitotic figures than placebo-treated animals (p<0.05). The maximal length of mitosis-free areas was 0.6 (0.6–0.9) mm in rats given indomethacin which is significantly larger than 0.4 (0.2–0.4) mm observed in controls (p<0.05). Indomethacin produced epithelial atrophy as shown by a significant reduction of the epithelial height observed in those rats compared to controls (p<0.05).The inhibition of cell proliferation observed in the oxyntic mucosa of rats treated with the cyclooxygenase blocker indicates that an important physiological role of endogenous prostaglandin is to maintain the proliferative activity of the epithelium at a high level.