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Roles of Fos in apoptosis of epithelia in the mouse male accessory sex organs and uterus were investigated using Fos-deficient mice. Normal 30- and 50-day-old and Fos-deficient 50-day-old male and female mice were castrated, and testosterone propionate and estradiol-17 beta were daily injected into male and female mice, respectively, for 5 days. An apoptotic index (a percentage of apoptotic cells) in the epithelium was examined from the day following the last injection (day 1) to day 8. The body weights and the weights of the ventral prostate (VP), coagulating gland (C), seminal vesicle (SV) and epididymis (Ep) and uterus of 50-day-castrated Fos-deficient mice on day 1 suggested that the development of these mice corresponded to that of 30-day-castrated normal mice at the most. The extents of apoptosis estimated by an apoptotic index in the VP, C, SV, Ep and uterus in 50-day-castrated Fos-deficient mice were comparable to those in 30-day-castrated normal mice. The extents of apoptosis in the SV, Ep and uterus in 30-day-castrated normal and 50-day-castrated Fos-deficient mice were similar to those in 50-day-castrated normal mice, while the extents of apoptosis in the VP and C in the former two groups of mice were less than those in the latter mice. The present results show that Fos-deficiency does not affect apoptosis in the SV, EP and uterus. However, the extents of apoptosis in the VP and C were less in 50-day-castrated Fos-deficient mice than in 50-day-castrated normal mice. This seems to be due to the retarded development of 50-day-castrated Fos-deficient mice, but not to a role of Fos in apoptosis.

Castration on days 0, 5, 10, 20, 40, and 60 caused increases in an apoptotic index (% of apoptotic cells) in seminal vesicle (SV) epithelium, peaking 1-3 days after castration. The peak apoptotic indices after castration on days 0, 5, 10, and 20 were significantly lower than peak apoptotic indices observed after castration on days 40 and 60. DNA extracted from mouse SVs 2 days after castration on days 0, 5, 10, and 60 showed a ladder pattern on agarose gel electrophoresis. The secretion of androgen by testes was confirmed by the growth retardation of the SVs after castration on days 0, 5, 10, and 20. It would appear that a proportion of SV epithelial cells dependent on testicular androgens for survival is smaller before day 20 than after day 20.

The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.

The effect of castration on apoptosis in the mouse epididymis during postnatal development was examined. The weight of the epididymis slowly increased from day 0 (day of birth) to day 20 after birth, followed by a rapid increase thereafter. Castration on days 0, 5, 10, 20, 30, 40 and 60 increased apoptotic indices (percentages of apoptotic cells) of epithelia of the caput (head), corpus (body), and cauda (tail) epididymis, their apoptotic indices reaching maximal levels on day 2 after castration with the exception of a maximal apoptotic index on day 4 in the tail after castration on day 60. The maximal levels of apoptotic indices of the head, body and tail after castration on days 0, 5, 10 and 20 were significantly lower than those after castration on days 40 and 60. DNAs extracted from the epididymides 2 days after castration on days 0, 5, 10 and 60 showed a ladder pattern on agarose gel electrophoresis, which is a characteristic of apoptosis. When testosterone propionate (10 microg/g body weight) was injected twice a day into mice which had been castrated on day 10, 30 or 60, the increases in apoptotic indices of the head, body and tail of the epididymis were completely inhibited. The weights of the paired epididymides 6 days after castration on days 0, 5, 10, 20, 30, 40 and 60 were significantly lower than those of sham-operated mice, indicating the secretion of androgen by the testes from birth to adulthood. The present results indicated that androgen deprivation caused by castration induces apoptosis in the epithelium of the epididymis of mice from birth to adulthood, and suggested that a proportion of epithelial cells, the survival of which is dependent on the testes, is smaller in the epididymides during a slow growth stage than in the epididymides after this stage.

The role of the Fas ligand-Fas system in castration-induced apoptosis in the epithelia of the ventral prostate (VP), seminal vesicle (SV), coagulating gland (CG) and epididymis (Ep) was investigated using lpr/lpr, and gld/gld mutant mice which are deficient in Fas and Fas ligand, respectively. The degree of apoptosis in the epithelium was quantitatively estimated by an apoptotic index (a percentage of apoptotic cells). The weights (mg/10 g body weight) of the VP, SV, CG and Ep of lpr/lpr and gld/gld mice were similar to those of normal +/+ mice and castration decreased the weights of the VP, SV, CG and Ep in these three kinds of mice to similar levels. Castration also increased the apoptotic indices in these organs reaching maximum on days 2-6 after castration. There was no significant difference in the apoptotic index of these organs among +/+, lpr/lpr and gld/gld mice on days 0-8 after castration. Agarose gel electrophoresis of DNAs extracted from the VP, SV, CG and Ep of +/+, lpr/lpr and gld/gld mice on day 4 after castration showed a ladder pattern. The present results suggest that the Fas ligand-Fas system plays little role in castration-induced apoptosis in the mouse male accessory sex organs such as the VP, SV, CG and Ep.

Background: Antigen receptor ligation induces apoptosis of B lymphocytes, but the molecular mechanisms underlying induction of apoptosis remain unclear, although the growing family of IL-1beta-converting enzyme cysteine proteases (caspases) are recognized to be major effectors of cellular death., Objective: We sought to delineate and compare the rescue of B-cell apoptosis through CD40 ligand-CD40 interaction and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A in human B cells., Methods: By using tonsillar B cells and the B-lymphoblastoid cell line Ramos, rescue from B-cell apoptosis was compared, as were signaling pathways after activation of cells through CD40 and the adenosine A2 receptor., Results: Both CD40 ligand-CD40 interaction and activation of intracellular cAMP rescue B cells from apoptosis after antigen receptor ligation. Although these pathways do not overlap, they converge by preventing the anti-IgM-induced activation of CPP32 (caspase 3), a member of the IL-1beta-converting enzyme protease family., Conclusion: These data indicate that the cAMP-protein kinase A-dependent and CD40-signaling pathways regulate B-cell survival and converge at a common point, the inhibition of antigen receptor-induced activation of caspases.

A combination of in vitro embryonic stem (ES) cell differentiation and targeted gene disruption has defined complex regulatory events underlying oxidative stress-induced cardiac apoptosis, a model of postischemic reperfusion injury of myocardium. ES cell-derived cardiac myocytes (ESCM) having targeted disruption of the MEKK1 gene were extremely sensitive, relative to wild-type ESCM, to hydrogen peroxide-induced apoptosis. In response to oxidative stress, MEKK1-/- ESCM failed to activate c-Jun kinase (JNK) but did activate p38 kinase similar to that observed in wild-type ESCM. The increased apoptosis was mediated through enhanced tumor necrosis factor alpha production, a response that was positively and negatively regulated by p38 and the MEKK1-JNK pathway, respectively. Thus, MEKK1 functions in the survival of cardiac myocytes by inhibiting the production of a proapoptotic cytokine. MEKK1 regulation of the JNK pathway is a critical response for the protection against oxidative stress-induced apoptosis in cardiac myocytes.

A gonadotropin-releasing hormone agonist, leuprolide acetate, was administered every 4 weeks for treatment of rectovaginal endometriosis. Degrees of apoptosis (percentage of in situ deoxyribonucleic acid 3'-end-labeled cells) and cell proliferative activity (percentage of cells with immunostaining for proliferating cell protein Ki-67) were examined in endometriotic glands of biopsy specimens taken before and during gonadotropin-releasing hormone agonist therapy. Gonadotropin-releasing hormone agonist induced apoptosis and suppressed cell proliferative activity in endometriotic glands.

Androgens affect many different target organs within the male reproductive tract to stimulate their development and secretory cytodifferentiation, and to maintain structure and function in adulthood. Castration causes regression of these organs via apoptosis. However, not all organs of the reproductive tract are equally sensitive to androgen withdrawal. The effects of castration on the mouse seminal vesicles (SVs) and bulbourethral glands (BUGs) were compared in terms of protein and DNA contents, epithelial apoptosis, and proliferative response of epithelial cells to androgen. Castration induced similar, marked decreases in protein contents in the SV and BUG by 2 days after castration which reached a minimum at 16 days post castration. Both organs underwent a decrease in DNA content, but the kinetics of this decline differed. In the SV, DNA content was significantly decreased by 4 days whereas in the BUG this did not occur until 16 days post castration. By day 16 both organs had regressed to roughly the same degree. The apoptotic index in the epithelium reflected this difference in timing as well. Apoptotic index of the SV epithelium was highest on day 3 after castration and declined thereafter. On the other hand, the apoptotic index in the BUG didn't begin to increase until 7 days after castration and became maximal on day 12. Daily injections of testosterone propionate (TP) from day 8, 16, or 30 after castration all increased epithelial labelling index in the SVs to a similar degree. However, the TP-induced increase in the epithelial labelling index in the BUG beginning on day 8 after castration was considerably less than that in BUGs receiving TP treatment from day 16 or 30 after castration. Thus, the proliferative response of the epithelium depended upon prior apoptosis in the gland, with the timing being delayed in the BUG as compared with the SV. The present results indicate that castration induces epithelial apoptosis and reduction in glandular DNA content considerably later in the BUG than in the SV though reduction in protein content in the BUG fell simultaneously with that in the SV.

Background: To clarify intranuclear apoptotic changes, we have investigated chromatin organization in apoptotic nuclei of castrated rat prostatic cells by a quick-freezing and deep-etching (QF-DE) method., Methods: The ventral prostates taken from intact and castrated adult male rats were investigated by light microscopy, in situ end-labeling (ISEL) technique, conventional electron microscopy, and the QF-DE method., Results: In control nuclei, the chromatin fibers were uniformly distributed and formed a network structure. In apoptotic nuclei, destruction of such chromatin networks was detected, which was clearly seen by the QF-DE method. Although it first appeared spotty in the apoptotic nucleus, definite destruction of the intranuclear network occurred in the nuclear center at later stages, and broken fibrous structures were condensed along the nuclear margin. The ISEL technique was applied to the QF-DE method. Localization of damaged DNA fragments could three-dimensionally be detected on replica membranes., Conclusions: Intranuclear chromatin organization in apoptotic cell death of rat prostates was observed by the QF-DE method. We could examine early-stage apoptotic nuclei at an electron microscopic level, which would not be clarified by other conventional methods.

Background: Information concerning the mechanisms underlying recovery from hydronephrosis is limited. The frequency of apoptosis during healing from hydronephrosis was studied using a rat kidney model., Methods: The presence of apoptosis was studied using an in situ DNA 3' end labeling method, electron microscopy, and agarose gel electrophoresis., Results: The degree of apoptosis in both the medulla and cortex gradually increased during ureteral obstruction as shown by in situ DNA 3' end labeling. Release of the ureteral obstruction resulted in a further increase in the degree of apoptosis in the medulla and cortex. The increase in apoptosis in the medulla was transient and lasted for only 4 days following release, while that in the cortex continued for at least 3 weeks. Apoptosis in the glomerulus was not observed. Electron microscopy revealed cells with aggregated chromatin in compact granular masses that abutted the nuclear membrane. Following release of ureteral obstruction, DNA fragmentation characteristic of apoptosis was visible on agarose gel electrophoresis., Conclusion: These results suggest that apoptosis is involved in post-obstructive tubular damage in the rat kidney.

Cell proliferation and apoptosis in uterine leiomyoma were investigated during therapy with GnRH agonist (GnRHa). Patients with uterine leiomyomas were injected with 3.75 mg GnRHa (depot leuprolide acetate) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of GnRHa therapy. Tissue sections of leiomyomas from these patients and from control patients (control patients received no GnRHa therapy) were stained with the Ki-67 antibody or by an in situ DNA 3'-end labeling method, and numbers of Ki-67 immunostained cells and DNA 3'-end-labeled cells per cm2 were examined as indices of cell proliferation and apoptosis, respectively. The number of Ki-67 immunostained cells/cm2 in leiomyomas at the 2nd week of the GnRHa therapy was comparable with that of control patients. However, it decreased to a level less than one forth that of control patients at the 4th week, and it remained at similar low levels at the 8th, 12th, and 16th week. The number of DNA 3'-end-labeled cells/cm2 in leiomyomas of control patients and in leiomyomas at the 2nd, 8th, 12th, and 16th weeks of GnRHa therapy were at low levels but, at the 4th week, was at an extremely high level (about 5 times more than that of control patients). The present results indicate that GnRHa therapy suppresses cell proliferation and causes a transient increase in apoptosis in uterine leiomyomas.

Background: Tissue samples from patients with prostatic adenocarcinoma (PCA) were examined for relationship between apoptosis and expression of Ki-67 and bcl-2 protein., Materials and Methods: Prostatic tissue was obtained from 29 prostatic cancer patients with no prior therapy. The number of apoptotic cells (apoptotic index; AI) and the percentage of Ki-67 stained cells (Ki-67 labeling index; LI) were compared with the expression of bcl-2 which was evaluated by the proportion and intensity of tumor cell immunostaining., Results: Both AI and LI of moderately and poorly differentiated PCAs were significantly higher than those of well differentiated PCAs. There was a significant correlation between AI and LI. Bcl-2 protein was expressed less in moderately and poorly differentiated PCAs than in well differentiated PCAs. Moreover, there was no correlation between the bcl-2 protein expression and the AI., Conclusions: The results suggest that, in PCAs, the degree of apoptosis is correlated with the cellular proliferation, and that expression of bcl-2 protein is not a critical factor that determines the degree of apoptosis.

The role of Fas in the apoptosis of testicular germ cells was investigated in BALB/c mice and Fas-deficient lpr/lpr mice. Spontaneous apoptosis of germ cells was observed in the testes of 40-day-old BALB/c mice, and experimentally induced cryptorchidism increased this apoptosis to such an extent that there was a decrease in the weight of the testis. Flow cytometry and immunohistochemistry using a Fas-specific monoclonal antibody demonstrated expression of Fas on germ cells including spermatogonia, spermatocytes, and spermatids. Furthermore, analysis by flow cytometry suggested that Fas expression on germ cells was increased following cryptorchidism. However, spontaneous and cryptorchidism-induced apoptosis of germ cells were also observed in 40-day-old Fas-deficient lpr/lpr mice. Moreover, testis weight also decreased following cryptorchidism in the mutant mice. The present results may indicate that the expression of Fas on germ cells does not correlate with spontaneous apoptosis or apoptosis induced by cryptorchidism. However, on the contrary, this study shows that Fas are partly involved in cryptorchidism-induced apoptosis, because the decrease in testis weight of lpr/lpr mice was less than that in BALB/c mice.

The immunosuppressant rapamycin (RAP) potentiated apoptosis of the murine T lymphoblastoid cell line S49 induced by dexamethasone (DEX), while RAP by itself did not induce apoptosis of the cells. FK506, in contrast, had no effect on DEX-induced apoptosis; moreover, an excess of FK506 reversed the potentiation of apoptosis by RAP, indicating that RAP exerts its effects through binding to FKBP. Both RAP and FK506 enhanced the MMTV promoter activity by dexamethasone, suggesting that the potentiation of apoptosis is not likely explained by the selective enhancement of transcriptional activity of the glucocorticoid receptor. Of interest, the basal activity of c-Jun kinase (JNK), whose activation has been recently suggested to be involved in cell survival signals in lymphocytes, was reduced by RAP in S49 cells. The reduction of JNK activity by RAP was reversed by the addition of an excess of FK506. In summary, we demonstrate for the first time that RAP has the ability to inhibit JNK activity in lymphocytes where the drug enhances apoptosis.

To examine the roles played by transforming growth factors (TGF)-beta1, -beta2, -beta3, and TGF-beta type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 microg estradiol-17beta (E2) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E2 pellets were removed, but administration of progesterone (P), 5alpha-dihydrotestosterone (DHT), or continued implantation of E2 pellets suppressed this increase. Levels of mRNAs of TGF-beta1, -beta2, and -beta3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-beta type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E2, P, and DHT markedly decreased the level of TGF-beta type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-beta1, -beta2, -beta3 and TGF-beta type II receptor were localized to the epithelium. Exogenous administration of TGF-beta1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-betas can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-betas, as demonstrated by an increase in TGF-beta type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-betas do not appear sufficient to induce apoptosis.

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), we investigated the effects of these two cytokines on death of luteal cells in vitro., Methods: Mouse luteal cells were cultured in serum-free medium with TNF-alpha at 0, 500, 1,000, 3,000, or 5,000 U/ml in the presence or absence of IFN-gamma at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3' end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy., Results: On day 3 of culture, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500-5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-gamma (1,000 U/ml) and TNF-alpha (5,000 U/ml) did. On day 6, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-alpha alone at 5,000 U/ml did, and combinations of IFN-gamma and TNF-alpha at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-alpha. On days 3-6 of culture, combinations of IFN-gamma and TNF-alpha that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis., Conclusions: The presence of IFN-gamma modulates the ability of TNF-alpha to induce a reduction in the number of viable cells, although TNF-alpha alone at high concentrations can induce a reduction in the number of viable cells.

Male Fischer 344 rats weighing 80-90 g were fed on a copper-depleted diet supplemented with 0.6% triethylenetetramine tetrahydrochloride (a copper chelator), and the death of pancreatic acinar cells of these rats was investigated morphologically and biochemically. The weight of the pancreas of these rats decreased from 3 weeks after feeding, and concomitantly the percentage of dead acinar cells increased to the maximum in about the 5th week and decreased subsequently. These dead acinar cells showed light microscopic and electron microscopic characteristics of apoptosis. Furthermore, the electrophoretic pattern of DNAs extracted from the pancreas having many dead acinar cells showed a ladder-like distribution, characteristic of apoptosis. The present results indicate that feeding of rats on a copper-depleted diet supplemented with a copper chelator results in apoptosis of acinar cells of the pancreas.

Female newborn mice were given daily injections of estradiol-17 beta (E2; 25 micrograms/mouse/day) for 4 days from the day of birth, and uterine cell death after this E2 priming was investigated by examining the apoptotic index (percentage of apoptotic cells), and the retention of 3H-radioactivity incorporated into epithelial or stromal DNAs after injection of [3H]thymidine into the mice on the day after birth. With injections of vehicle only after E2 priming, the apoptotic index of the uterine epithelium increased markedly, being maximal on day 4 of injections, and the 3H-radioactivity retained in the epithelium decreased rapidly. Agarose gel electrophoresis of uterine epithelial DNAs on day 4 of injections showed a ladder pattern, characteristic of apoptotic cell death. However, daily injections of E2 (7.2 micrograms/g body wt) completely inhibited the increase in the apoptotic index and the loss of 3H-radioactivity in the epithelium. Daily injections of progesterone (80 micrograms/g body wt), 5 alpha-dihydrotestosterone (DHT; 8 micrograms/g body wt), and dexamethasone (2 micrograms/g body wt) also inhibited both parameters, although not completely. The inhibitory effects of DHT and progesterone were abolished by the antiandrogen, flutamide and antiprogesterone, RU 486, respectively. In contrast, no apoptotic cells and no loss of 3H-radioactivity were found in the stroma for any treatment after E2 priming. The present results suggest that discontinuation of estrogen stimulation results in apoptotic cell death in the uterine epithelium of neonatal mice, but not in the stroma, and that estrogen, progesterone, DHT and dexamethasone inhibit cell death of uterine epithelium.