Prevention of death of axotomized hypoglossal neurones and promotion of regeneration by chitin grafting.

1. Chitin is known to promote skin wound healing. In this study, chitin, prepared from Zuwai crab shell, was used as a bridge between the proximal and distal stumps of cut hypoglossal nerves in shrews. We compared the effects of chitin on the regeneration of transected right hypoglossal nerve axons, with those of porcine dermis, bovine dermal aterocollagen, and autologous nerve bundles. 2. To assess the survival of neurones, the size of neuronal cell body, and number of motoneurones were determined in the absence of any bridged material and in the presence of porcine dermis, bovine dermal aterocollagen, chitin, or autologous nerve bundles as a bridge. 3. Our results revealed a significantly better outcome in chitin and autologous nerve bridged groups; the size of neuronal cell body and number of hypoglossal neurones were higher than in the other groups. Chitin also enhanced the regeneration of neurones; the number of horseradish peroxide positive neurones indicative of repaired axonal processes was significantly higher in chitin and autologous nerve-bridged groups than in other groups. 4. Our results demonstrated that the use of chitin sheet or autograft successfully prevented the death of severed neurones and promoted the regeneration of the lesioned nerve. Although the mechanisms underlying the effects of chitin are still unknown, chitin seems to be a potentially useful biocompatible material for nerve repair and regeneration.

Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey.

Hepatitis E virus (HEV) has been considered to be the major cause of enterically transmitted non-A, non-B hepatitis in developing countries. However, little is known about viral replication and localization in the liver. The aim of this study was to examine the distribution of HEV-infected cells in experimentally infected animals. Seven captured wild rhesus monkeys were inoculated intravenously with faecal extract derived from a Myanmar strain of HEV. Animals were killed at different time-points of clinical illness: during early infection, during prehepatitis with viral-like particles in bile, during acute hepatitis and during convalescence. Intrahepatic localization of HEV was analysed using non-isotopic thymine dimer in situ hybridization (NITDISH). Both plus and minus strands of HEV RNA were found in hepatocytes during the early infection period. Staining in the submembranous cytoplasmic region of hepatocytes was observed. In the prehepatitis period, both plus and minus strand HEV RNAs appeared in the canalicular side of isolated bile epithelial cells. Subsequently, HEV RNA became universally distributed in the cytoplasm of medium-size bile epithelial cells. After recovery, HEV RNA disappeared.

Role of apoptosis of thyrocytes in a rat model of goiter. A possible involvement of Fas system.

Apoptosis, a physiological process of cell death, may modulate the mass of the thyroid gland. We investigated the role of apoptosis and the possible involvement of Fas/Fas ligand (FasL) system in apoptosis during goiter formation and involution in a rat model of goiter. Rats were fed a low iodine diet and a goitrogen, 6-propyl-2-thiouracil, to induce goiter. Rats with goiter were then fed a high iodine diet to study the phase of involution. We examined the presence of apoptosis by electron microscopy (EM) and terminal deoxy-UTP nick end labeling (TUNEL). We also investigated the association between Fas and FasL expression and thyrocyte apoptosis using immunohistochemistry and Western blotting. To evaluate the proliferation of thyrocytes, proliferating cell nuclear antigen was examined immunohistochemically. The number of apoptotic cells increased during goiter formation and the early stage of involution, which were also associated with increased number of Fas-positive thyrocytes, and some of these cells contained TUNEL-positive nuclei. However, the expression of FasL was almost constant throughout the experiment. Proliferating cell nuclear antigen/TUNEL ratio markedly increased during goiter formation but decreased particularly during the late stage of goiter involution. Our results indicate that apoptosis of thyrocytes is a main factor of cell loss during goiter formation and involution and suggest that the Fas/FasL system is involved in the induction of apoptosis of these cells. Moreover, the delicate balance between apoptosis and cell proliferation may play an important role in the control of thyroid gland mass.

Expression of decay accelerating factor mRNA and complement C3 mRNA in human diseased kidney.

BACKGROUND: Decay accelerating factor (DAF), a product of mesangial cells in vitro, is expressed on the surface of cells and is a candidate for the focal suppression of complement activation. It is not clear at present whether the levels of expression of DAF and intrarenal C3 synthesis correlate with the level of tissue injury. METHODS: Immunohistochemistry for DAF and C3 and nonradioactive in situ hybridization with digoxigenin-labeled oligonucleotide probe for DAF and C3 mRNA were performed in 22 tissue samples of kidneys from patients with IgA nephropathy (IgAN), 6 with membranous nephropathy (MN), 6 with lupus nephritis (LN), and five normal kidneys. RESULTS: In the normal kidney, DAF was confined to the juxtaglomerular apparatus and little or no C3 was detected; however, a few glomerular cells were positive for DAF mRNA but no C3 mRNA positive cells were detected. In diseased kidneys, DAF and C3 as well as their mRNAs were detected in mesangial cells, tubular cells and infiltrating cells. Glomerular epithelial cells and Bowman's capsule cells contained little or no DAF and C3 but were positive for their mRNAs. The mean percentages of mesangial cells positive for DAF and C3 mRNAs were 49.3 +/- 11.5% and 50.7 +/- 10.3% in IgAN, and 17.0 +/- 6.3% and 19.4 +/- 9.0% in MN, respectively. The percentage of mesangial cells positive for DAF and C3 mRNAs among intraglomerular cells correlated positively with the degree of mesangial proliferation and glomerular sclerosis in IgAN. In contrast, in LN the percentage of glomerular cells positive for DAF mRNA correlated negatively with the degree of glomerular injury, while the percentage of cells positive for C3 mRNA did not change with the progression of the disease. The ratio of C3 mRNA/DAF mRNA of glomerular cells correlated with the degree of glomerular injury in both IgAN and LN. In the tubulointerstitium, the percentage of cells expressing mRNA, and C3 mRNA/DAF mRNA radio correlated with the degree of tubular atrophy and interstitial broadening in both IgAN and LN. CONCLUSIONS: We conclude that DAF and C3 mRNAs are synthesized in human diseased kidneys, and that a balance between locally synthesized DAF and C3 may be important in the progression of glomerulonephritis.

Activation of bullous pemphigoid antigen gene in mouse ear epidermis by ultraviolet radiation.

Bullous pemphigoid (BP) is an autoimmune blistering disease and is a photoaggravated dermatosis, but the mechanism of the aggravation is still unknown. Since damage to DNA initiates transcription of some genes, we investigated in epidermis of mouse ears the relationship between DNA damage by ultraviolet (UV) radiation and BP antigen (BP-Ag) gene activation. For this, albino male mice were irradiated with 254 nm wavelength UV for a total dose of 500 J m-2. At fixed times (0.5, 2, 24, 48 and 72 h) post-UV irradiation, mouse ears were cut off, frozen and sectioned. In the sections, it was found that immunohistochemically detectable pyrimidine dimers were observed in nuclei of all epidermal cells at 0.5 h that were almost repaired by 72 h; a frequency of single strand breaks in DNA detected by in situ nick translation started to increase in nuclei of all epidermal cell layers at 0.5 h and the increase continued up to 24 h; mRNA for BP-Ag localized by non-radioactive in situ hybridization appeared in nuclei of basal cells at 0.5 h and in both nuclei and cytoplasm at 2 h; and immunoreactive BP-Ag started to increase in the basal cell cytoplasm and in the basement membrane zone at 2 h. BP-Ag started to accumulate in the basement membrane zone at 2 h. It is suggested that UV radiation increased BP-Ag synthesis through BP-Ag synthesis through BP-Ag gene activation and that this reaction is a factor which aggravates BP following UV irradiation in BP patients.

Autonomous cell death of mouse male germ cells during fetal and postnatal period.

Germ cell degeneration is common in mammalian testes during the developmental as well as the adult period. To investigate the extent and mechanisms of male germ cell death during fetal and neonatal life, the testes of mice at various fetal and postnatal ages extending from 13 days of gestation to 7 wk after birth were examined by electron microscopy and/or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Electron microscopy revealed that the number of cells with typical features of spermatogenic cell apoptosis was highest at 13 days of gestation, coinciding with the time of immigration of primordial germ cells into gonads. A second peak was observed around 10-13 days after birth when the first wave of spermatogenesis had started and active spermatogonial proliferation was present. Surprisingly, we found a significant number of dying cells around birth, which exhibited morphological features of necrotic death. In agreement with the results of electron microscopy, TUNEL staining revealed that the dying germ cells present around birth were TUNEL negative, while positive nuclei were abundant in the lumen of seminiferous tubules of testes of 10- to 13-day-old mice. To investigate the mechanisms of induction of germ cell death, we examined the expression of Fas antigen immunohistochemically using rabbit antiserum raised against synthetic peptides for part of mouse Fas antigen. We found that among various developmental stages investigated, positive immunostaining for Fas antigen was present between 17 days of gestation and 1 day after birth, with the most intensive staining occurring on 17 days of gestation. Therefore, Fas-induced pathways may be implicated in embryonic male germ cell death, not prepubertal spermatogenic cell death.

Cellular expression of beta-microseminoprotein (beta-MSP) mRNA and its protein in untreated prostate cancer.

BACKGROUND: Previous studies have shown that beta-microseminoprotein (beta-MSP) may be used as a diagnostic marker for prostate cancer. However, the level of expression of beta-MSP in prostate cancer detected by immunohistochemistry (IHC) has varied from one study to another. METHODS: We analyzed the expression of both beta-MSP mRNA and its protein in a large sample of prostate tumors from 104 patients with untreated prostate cancer, using both nonradioactive in situ hybridization (ISH) and IHC. RESULTS: Our results showed that 72 and 96 of 104 specimens were negative for beta-MSP mRNA (69.2%) and beta-MSP (92.3%), respectively. Furthermore, a reduced expression of both beta-MSP mRNA and its protein was detected in all malignant epithelial tissues compared with benign epithelia. Not all malignant tissue samples negative for beta-MSP mRNA were negative for beta-MSP (6.7%), and vice versa (29.8%). Other tissue samples were either negative for both (62.5%) or positive for both (1.0%). CONCLUSIONS: Our results showed a lower level of expression of beta-MSP in prostate cancer tissue, compared with benign prostate tissue. This phenomenon may be mainly due to the presence of reduced levels of beta-MSP mRNA.

Induction of apoptosis in ischemia-reperfusion model of mouse kidney: possible involvement of Fas.

Although ischemia-reperfusion of mouse kidney is known to cause severe renal failure due to tubular cell death, the exact cellular mechanism responsible for this phenomenon is not clear. To investigate the spatial and temporal development of renal cell death and the role of Fas/APO-1/CD95 (Fas) in this process, the left renal vessels were occluded in a group of mice for 30, 60, or 120 min followed by reperfusion for 24 h (n = 4 for each group). Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of multiples of 180 to 200 bp, considered the hallmark of apoptosis. The intensity of the bands increased proportionately with the duration of ischemia. Histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling showed the presence of nuclei with DNA double-strand breaks specifically in distal renal tubules of the outer medulla. The presence of apoptosis was also confirmed by electron microscopy. Analysis of total RNA by Northern blotting revealed one appropriate-sized band for Fas mRNA in the normal kidney, which intensified in the ischemia-reperfused kidney. Moreover, nonradioactive in situ hybridization revealed that distal renal tubular epithelial cells were positive for Fas mRNA in the outer medulla. Fas antigen was also localized to the renal tubular epithelial cells of the outer medulla by immunohistochemistry. The number of apoptotic cells in the ischemia-reperfusion kidney of the lpr/lpr mouse was low. These findings strongly indicate that ischemia-reperfusion of the kidney induces apoptosis of a specific area of tubular epithelial cells in the outer medulla through the Fas system.

Differential expression of IFN-gamma, IL-4, IL-10, and IL-1beta mRNAs in decalcified tissue sections of mouse lipopolysaccharide-induced periodontitis mandibles assessed by in situ hybridization.

To examine the role of T cell subgroups, Th1 and Th2, in the development of periodontitis, the expression of various cytokines was investigated in a mouse model of alveolar bone resorption using in situ hybridization (ISH) with digoxigenin-labeled oligonucleotides. When mice received repetitive injections with Escherichia coli lipopolysaccharide into the gingiva every 48 h, alveolar bone resorption was detectable after the fourth injection, reaching a maximum after the 13th injection. For the best performance of ISH, we first had to choose a decalcification protocol. Among various decalcification protocols, 10% EDTA (4 degrees C, 5-6 days) was the best for 28S rRNA staining. Positive cells for transcripts of interferon-gamma (Th1 product) were detected after the fourth injection, reaching a maximum after the tenth injection. A similar pattern was obtained for interleukin (IL)-10 mRNA (Th2 product) and IL-1beta, while the positive cell number reached a maximum after the 13th and 10th injections, respectively. The number of IL-4 mRNA (Th2 product)-positive cells remained low till the tenth injection, but increased thereafter. Consequently, we found that the population change from Th1 to Th2 in the inflammation site correlated with the transition from gingivitis to periodontitis, indicating differential roles of T cell subgroups in the development of periodontitis.

Frequency of apoptosis relates inversely to invasiveness and metastatic activity in human colorectal cancer.

The frequency of apoptosis was determined in 102 cases of human colorectal cancer. The results were correlated with the frequency of cell proliferation and with clinicopathological characteristics such as degree of differentiation, invasiveness and metastasis. As a marker of apoptosis, intranuclear DNA strand breaks were localized with in situ nick translation (ISNT). As a marker of proliferation, proliferating cell nuclear antigen (PCNA) was localized immunohistochemically. The numbers of nuclei positive with ISNT and for PCNA per 1,000 nuclei on tissue sections were obtained. The labelling indices were compared with clinicopathological characteristics for each tumour. The ISNT labelling index of well differentiated colon carcinomas was higher than that of poorly differentiated carcinomas. Among similarly differentiated cancers, ISNT L.I. of colon carcinomas classified as Dukes A was higher than Dukes B/C, and L.I. of carcinomas which did not metastasize to lymph node or liver was higher than that of carcinomas which metastasized. The PCNA labelling index did not correlate with any of the clinicopathological characteristics or with the ISNT labelling index. The data suggest that apoptosis indices severe as a marker of tumour progression.

Intraglomerular C3 synthesis in rats with passive Heymann nephritis.

Passive Heymann nephritis (PHN), a model of human membranous nephropathy, is an immune-complex-mediated glomerulonephritis characterized by the presence of complement-dependent tissue injury. Recent studies have confirmed the synthesis of C3, involved in both the classical and alternative pathways of complement, in injured human and animal renal tissues. However, there is little clear information on the role of local C3 synthesis in the pathogenesis of nephritides such as PHN. In the present study, using nonradioactive in situ hybridization and semiquantitative reverse transcriptase polymerase chain reaction, we examined C3 synthesis in the kidney and its contribution to tissue injury in a rat model of PHN induced by the injection of polyclonal anti-gp330 antibody. C3 mRNA was localized in mesangial cells, glomerular epithelial cells, and cells of Bowman's capsule. During the early stages of PHN, C3 mRNA expression was detected in mesangial cells and glomerular epithelial cells, whereas such expression was limited to mesangial cells during the late stages of the disease. Focal, weak C3 mRNA expression was detected in tubular epithelial cells and occasionally in the interstitium. Semiquantitative polymerase chain reaction demonstrated that the level of C3 mRNA expression correlated with that of proteinuria. Our results suggest that renal cells synthesize C3 mRNA in PHN in a site-specific manner and that locally produced C3 is associated with the development of proteinuria in this model.

Fas and Fas ligand interaction is necessary for human osteoblast apoptosis.

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoylphorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.

Involvement of IL-4 in human glomerulonephritis: an in situ hybridization study of IL-4 mRNA and IL-4 receptor mRNA.

Interleukin-4 (IL-4) has been recently implicated in the pathogenesis of glomerulonephritis. However, the expression of IL-4 and IL-4 receptor (IL-4R) in human kidney has not been fully determined. Nonradioactive in situ hybridization was used to examine the expression of IL-4 mRNA and IL-4R mRNA in tissues from normal kidneys and specimens from a variety of human kidney diseases. In normal glomeruli, a few mesangial cells and cells of the Bowman's capsule weakly expressed IL-4 and IL-4R mRNA, whereas in diseased glomeruli both mRNA types were strongly expressed in resident glomerular cells, including mesangial cells, glomerular epithelial cells, and cells of the Bowman's capsule. The relationship between the expression of these mRNA and the degree of glomerular injury was different in different types of glomerulonephritis. In IgA nephropathy and non-IgA mesangial proliferative glomerulonephritis, IL-4 expression correlated positively with the degree of mesangial hypercellularity and extracellular matrix expansion. However, IL-4R expression was relatively constant. In contrast, the expression of IL-4 and IL-4R mRNA correlated negatively with the degree of glomerular injury in lupus nephritis. Coexpression and discordant expression of these mRNA forms were observed in individual cells. In tubulointerstitium with severe lesions, IL-4 mRNA and IL-4R mRNA were observed in atrophic tubules and some of the infiltrating cells and fibroblasts. The interstitial expression of these mRNA forms was similar in IgA nephropathy, non-IGA mesangial proliferative glomerulonephritis, and lupus nephritis and correlated positively with the degree of tubulo-interstitial changes. These results suggest that an autocrine and/or paracrine pathway of IL-4 is present in human diseased kidneys and that IL-4 may be involved in tissue injury in glomerulonephritis.

Expression of estrogen and progesterone receptors in endometrium and peritoneal endometriosis: an immunohistochemical and in situ hybridization study.

OBJECTIVE: To clarify the role of ovarian steroids in the development and progression of endometriosis, estrogen receptors (ERs) and progesterone receptors (PRs) were localized by immunohistochemistry, and ER messenger RNA (mRNA) was detected by in situ hybridization in the uterine endometrium and in normal and altered pelvic peritoneum. DESIGN: Retrospective and prospective study. SETTING: Nagasaki University School of Medicine, Nagasaki, Japan. PATIENT(S): A retrospective study of 61 formalin-fixed uterine endometria and normal and altered pelvic peritonea from patients suffering from various gynecologic diseases was conducted. In addition, in 22 fresh frozen tissue specimens, ER mRNA expression was evaluated prospectively. MAIN OUTCOME MEASURE(S): In formalin-fixed tissues, ER and PR were localized immunohistochemically. The results of immunohistochemical staining were scored from 0 to 4, depending on the signal intensity and frequency of positive cells. In fresh frozen specimens, ER mRNA expression was assessed by nonradioactive in situ hybridization using thymine-thymine dimerized oligonucleotide probes. RESULTS: The highest score of ERs and PRs was observed in the epithelial and stromal cells of the normal uterine endometrium at the early proliferative phase of the menstrual cycle. The ER and PR scores declined throughout the secretory phase. In typical endometriotic lesions, the ER and PR scores were constantly high independent of the menstrual cycle. The expression pattern of ER mRNA was mostly in parallel with that of ERs. In typical endometriosis, ERs and PRs were found in both glandular epithelial cells and their surrounding stromal cells. Expression of ER mRNA was found in typical endometriotic peritonea and in pelvic peritoneum with columnar epithelial cells, but not in normal pelvic peritoneum (mesothelium). Estrogen receptors and PRs were negative in mesothelium, but were positive in the nuclei of fibroblasts in the connective tissue. CONCLUSION(S): We demonstrated the expression of ERs, ER mRNA, and PRs in the columnar cells in pelvic peritonea and typical endometriosis, but not in normal mesothelium. These results suggest that endometriosis may originate from the columnar cells with ERs and PRs in the pelvic peritoneal lining.

Intraglomerular C3 synthesis in human kidney detected by in situ hybridization.

Complements 3 and 4 are known to be synthesized in diseased renal tissue and the mRNA of these complements has been demonstrated, using polymerase chain reaction, in renal biopsies from nephritic patients. However, the types of cells producing the complements in intact renal tissue have not been defined. To identify the renal cellular components involved in complement synthesis, we analyzed the expression of C3 mRNA in renal tissues from patients with immune-complex glomerulonephritis by a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide. Renal tissues from 15 patients with immunoglobulin (Ig) A nephropathy (IgAN), five with lupus nephritis (LN), and five with minimal change nephrotic syndrome (MCNS) were examined. Uninvolved portions of surgically removed kidney with tumors served as normal controls. C3 mRNA was detected in mesangial cells, glomerular epithelial cells, and Bowman's capsule in IgAN and LN. In the interstitium, some tubules and some infiltrating mononuclear cells were positively stained for C3 mRNA. C3 mRNA was not detected in MCNS and control tissues. Our results confirm that the glomerular resident cells can synthesize C3 in immune-mediated glomerulonephritis and suggest that locally synthesized complement may be involved in tissue injury in glomerulonephritis.

Apoptosis of crypt epithelial cells in ulcerative colitis.

In the colon of ulcerative colitis (UC) patients, apoptotic bodies have been recognized in routine histopathological preparations. To investigate the extent of the apoptosis, colonic biopsies were examined from involved and uninvolved areas of untreated active UC and from normal areas in patients with colonic polyps, utilizing various markers of apoptosis. The markers included DNA breaks detected by TUNEL, Fas (CD95/APO-1) and Fas ligand (Fas-L) localized by immunohistochemistry, electron microscopic features of apoptosis, and laddering of extracted DNA. Apoptosis marker positive cells were found mainly on the luminal epithelium of the normal colon and were present in active UC in crypts of involved and uninvolved areas of the colon, in addition to the luminal epithelium. The DNA extracted from active UC colon electrophoresed as a ladder. These findings suggest that the loss of epithelial cells in active UC occurs mainly by apoptosis in crypts of involved and adjacent uninvolved areas and that the Fas/Fas-L interaction is a mediator of the apoptosis.

[Detection of apoptosis and expression of Fas antigen in human colorectal cancer].

The occurrence of apoptosis in formalin-fixed, paraffin-embedded human colorectal cancer tissues was investigated at a cellular level by in situ nick translation (ISNT). Then the frequency of ISNT-positive nuclei was compared with the proliferative activity assessed by proliferating cell nuclear antigen (PCNA) labeling index, and with the incidence of Fas positive cells examined immunohistochemically with the incidence of Fas positive cells examined immunohistochemically with rabbit anti-Fas serum. As a result, although no significant correlation between the frequency of apoptosis and the proliferative activity was observed, a balance between them may explain a variety of growth rates of colorectal cancers. As for Fas expression, about 33% of the colorectal cancers were more or less positive for Fas antigen.

[Expression of Fas and Fas-ligand in epithelium of ulcerative colitis].

Ulcerative colitis (UC) is characterized by the loss of epithelium and inflammation of lamina propria. In normal colon, epithelial cells are eliminated by apoptosis at the luminal surface. The apoptotic cells recognized by their typical morphology and the presence of DNA breaks are also accompanied by Fas and Fas-ligand. It is thought when Fas-ligand bound the Fas, the cells bearing Fas will enter a path of apoptosis. On the other hand, in inflamed areas of UC and adjacent un-inflamed areas, the apoptosis of epithelial cells are recognized anywhere along crypts. These findings suggest that unscheduled apoptosis in the UC crypts takes place and the apoptosis is mediated by a auto- or paracrine Fas/Fas-ligand interaction.

Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia.

Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.

Recent advances in molecular histochemical techniques: in situ hybridization and southwestern histochemistry.

Over the past decade, considerable efforts to understand the states of specific gene expression at cellular and/or subcellular levels have been made. For this particular purpose, nonradioactive in situ hybridization to localize mRNAs has been developed and improved substantially, and it is now recognized as a powerful, established light-microscopical technique. In this review, we focus on the recent advances in the technological aspects of nonradioactive in situ hybridization including the use of synthetic oligodeoxynucleotide probes, the progress in analysis of signals, and the application to electron microscopy. Also, southwestern histochemistry, a relatively new method of localizing transcription regulatory proteins by utilizing haptenized DNA with responsive element sequences is described. Then we discuss what we can see by combining these molecular histochemical methods which were brought about by the merger of molecular biology and structural biology.