Epithelial cell proliferation and apoptosis in the developing murine palatal rugae

Epithelial cell proliferation and apoptosis during morphogenesis of the murine palatal rugae (PR) were examined histochemically by using anti-bromodeoxyuridine (BrdU) and the terminal deoxynucleotidyl transferase-mediated UTP nick-end-labelling (TUNEL) technique. Formation of the PR rudiment was observed as an epithelial placode in fetuses at 12.5 days post-coitus (dpc). During the PR formation, BrdU-positive cells were detected mainly in the epithelium of the interplacode and interprotruding areas in fetuses administered BrdU maternally at 2 h before killing. TUNEL-positive cells were detected only at the epithelial placode area in 12.5-14.5 dpc. At 16.5-18.5 dpc, the BrdU-positive cells were decreased in number in the epithelial cells at the interprotruding area of the PR. Only a few TUNEL-positive cells were observed in the protruding area of the PR at 16.5 dpc. These results suggest that cell proliferation and apoptosis in the palatal epithelium are involved spatiotemporally in the murine PR morphogenesis.     

Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar

The objective of this study was to determine the pathogenesis of experimental infection with a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the sites of viral replication and apoptosis in male gonads from infected boars for a period of 21 days after intranasal inoculation. Microscopically, hypospermatogenesis and abundant germ cell depletion and death were observed in the testes. Such germ cell death occurs by apoptosis, as determined by a characteristic histological patterns and evidence of massive DNA fragment detected in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction. PRRSV was detected in the testicular tissue of infected boars only. Viral nucleic acid was localized in spermatogonia, spermatocytes and spermatids but not in the vesicular and bulbourethral gland. In serial sections, PRRSV-positive cells did not co-localized with apoptotic cells. TUNEL-positive apoptotic cells were more numerous than PRRSV-positive cells in testicular sections. The present study demonstrated that type 1 PRRSV infects the spermatogonia and their progeny, and induces apoptosis in these germ cells.     

Apoptosis in feline panleukopenia and canine parvovirus enteritis

Tissue samples of cats and dogs with panleukopenia and parvovirus enteritis, respectively, were examined for the presence of viral antigen-positive cells and apoptotic cells by immunohistochemistry and by TUNEL assay (Terminal Transferase-Mediated dUTP Nick End Labelling). Compared to control animals, infected cats and dogs generally had more TUNEL-positive cells. Cell types positive for parvovirus antigen, for example digestive tract epithelial and mesenchymal cells, and lymphocytes and macrophages in lymphoid tissues were also positive for TUNEL signals. Occasionally, TUNEL signal and viral antigen were present in the same tissue areas, suggesting a direct viral trigger of apoptosis. More frequently, however, there was no complete overlap of antigen and TUNEL-positive areas. The results of this study indicate that apoptotic cell death contributes significantly to the widespread tissue damage of parvovirus infection in cats and dogs.     

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

Developmental changes in cell proliferation and apoptosis in the normal duck thymus

Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.     

Assessment of apoptosis in epidermal lamellar cells in clinically normal horses and those with laminitis

OBJECTIVE: To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis. SAMPLE POPULATION: Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis). PROCEDURE: Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis. RESULTS: The number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUNEL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14. CONCLUSIONS AND CLINICAL RELEVANCE: The large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated.     

Relationship of Cell Proliferating Marker Expressions with PGE(2) Receptors in Regenerating Rat Renal Tubules after Cisplatin Injection

Cisplatin, an anticancer drug, is well known to have nephrotoxicity as an adverse effect. We investigated the expressions of cell cycle markers and prostaglandin E(2) (PGE(2)) receptors (EP) in the affected renal tubules in rats injected with a single dose (6 mg/kg body weight) of cisplatin. On days 1-3 after dosing, the affected renal epithelial cells were almost desquamated, showing necrosis. On day 5 onwards, the renal tubules were rimmed by flattened or cuboidal epithelial cells with basophilic cytoplasm; BrdU-immunopositive cells began to significantly increase, indicating regeneration. Simultaneously, TUNEL-positive apoptotic cells were also seen. On days 1-5, cyclin D1-immunopositive cells were decreased with an increased expression in p21 mRNA, indicating G(1) arrest in the cell cycle. The affected renal epithelial cells began to react to EP4 receptor, but not to EP2 receptor. Some EP4 receptor-reacting epithelial cells gave a positive reaction to BrdU or cyclin D1. Collectively, the affected renal tubules underwent various alterations such as necrosis, apoptosis, regeneration and G(1) arrest; the aspects might be influenced by endogenous PGE(2) through EP4 receptor.     

miR-142-3p对人乳腺上皮细胞MCF-10A增殖及乳蛋白合成的影响

MicroRNAs(miRNAs)是一类长度约为22 nt的非编码的调控性小RNA,在诸多生命活动中发挥重要作用,如参与调控细胞的增殖、分化、凋亡及肿瘤的发生发展。本试验应用脂质体转染技术抑制miR-142-3p在人乳腺上皮细胞的表达。试验采用实时荧光定量PCR、Western blotting、细胞增殖分析等技术,探索miR-142-3p对人乳腺上皮细胞增殖及乳蛋白合成的影响。结果显示,miR-142-3p沉默后,催乳素受体(prolactin receptor,PRLR)蛋白表达增强,同时,相关通路蛋白AKT、mTOR、STAT5、cyclinD1表达量均增加,细胞增殖能力增强。结果表明,在人乳腺上皮细胞中,miR-142-3p的沉默使PRLR蛋白表达量升高,通过调控AKT、mTOR、STAT5、cyclinD1相关通路蛋白而促进乳蛋白质的合成和乳腺上皮细胞的增殖。     

Effect of miR-142-3p on Cell Proliferation of Human Mammary Epithelial Cells MCF-10A and Synthesis of Milk Protein

MicroRNAs (miRNAs) are a group of small,non-coding RNA molecules about 22 nucleotides to regulate a wide variety of important biological processes,including cell proliferation,differentiation,apoptosis as well as the progression of tumors.Liposome transfection was used to detect the expression of miR-142-3p in human mammary epithelial cells,the effects of miR-142-3p on the cell proliferation,apoptosis and milk protein synthesis were detected by Real-time PCR,Western blotting,cell proliferation analysis.The results indicated that after miR-142-3p being silenced,prolactin receptor (PRLR) protein was increased,at the same time the expressions of related pathways protein AKT,mTOR,STAT5 and cyclinD1 were increased,the ability of cell proliferation was increased.The results suggested that in human mammary epithelial cells,the silence of miR-142-3p could increase the expression of PRLR protein,miR-142-3p could promote the synthesis of milk protein and increase the proliferation of mammary epithelial cells by regulating related pathways proteins AKT,mTOR,STAT5 and cyclinD1.     

羊驼睾丸细胞凋亡及凋亡相关蛋白的定位

本研究旨在观察羊驼睾丸的出生后发育和精子发生过程中的细胞凋亡及凋亡相关蛋白Bcl2和Caspase3 的定位.取材新生、12月龄和24月龄羊驼的睾丸,用TUNEL法检测睾丸发育和精子发生过程的细胞凋亡,用免疫组织化学技术检测凋亡相关蛋白Bcl2和Caspase3在羊驼出生后发育和精子发生过程中的定位.结果显示在新生羊驼睾丸未检测到TUNEL阳性细胞,Caspase3和Bcl2表达于间质细胞,提示在新生期凋亡蛋白参与间质细胞凋亡的调节,为曲精小管的发育提供空间;12月龄羊驼睾丸TUNEL阳性细胞定位于曲精小管中央部分,Caspase3 和Bcl2定位于间质细胞和曲精小管中央生殖细胞,提示在青春期(12月龄)羊驼睾丸,细胞凋亡和凋亡相关蛋白参与曲精小管管腔形成的调节;24月龄羊驼睾丸TUNEL阳性细胞定位于精原细胞、精母细胞和精子细胞,Caspase3 和Bcl2定位于间质细胞和各个发育阶段的生精细胞,Caspase3阳性细胞在精原细胞最高,向精母细胞和精子细胞逐渐减少,Bcl2在精原细胞弱阳性表达,在血睾屏障以内的曲精小管近腔室部分呈弥散性强阳性表达,提示在性成熟(24月龄)羊驼睾丸精子发生过程中,细胞凋亡主要发生于精原细胞和早期精母细胞,Bcl2可能抑制精母细胞之后生殖细胞的凋亡.结果提示在羊驼睾丸出生后发育和精子发生过程中存在细胞凋亡现象;凋亡蛋白Caspase3和Bcl2参与羊驼睾丸发育和精子发生过程中细胞凋亡的调节.     

Expression of Survivin,Bcl‐2 and Bax Proteins in the Domestic Cat (Felis catus) Endometrium During the Oestrus Cycle

Apoptosis has been shown to be an important regulator of endometrium function. To clarify the regulation of apoptosis in the cat endometrium during the normal oestrus cycle, the expressions of the apoptosis‐related proteins (Bcl‐2 and Bax) and their correlation to the inhibitor of apoptosis protein Survivin were analysed using immunohistochemistry. The TUNEL technique (TdT‐mediated dUTP nick end labelling) was also used to detect DNA fragmentation characteristic of apoptotic cells. The results demonstrated that TUNEL labelling is not effective for the detection of apoptosis in cat endometrium. Survivin was expressed in the luminal and glandular epithelial cells of cat endometrium during all phases of the oestrus cycle. Survivin was localized in both the cytoplasm and nuclei of superficial and deep uterine gland cells during the luteal phase, while only cytoplasmic staining was observed during the follicular and anoestrus phases. Bax immunoreactivity in the cytoplasm of luminal and glandular epithelial cells as well as the smooth muscle cells of blood vessels was weak in the anoestrus phase. Compared with anoestrus, the intensity of Bax immunostaining was moderate in the follicular phase and increased dramatically in the luteal phase. Bcl‐2 immunostaining in the cytoplasm of luminal and glandular epithelial cells was moderate in the anoestrus phase. During the early follicular phase, cytoplasmic Bcl‐2 immunostaining was detected mostly in glandular epithelial cells. In the mid‐follicular phase, in glands, the amount of Bcl‐2 protein increased progressively from the superficial to the deep layer. In contrast, the expression of Bcl‐2 decreased in the secretory phase, being very low or absent in the mid‐ and late luteal phases. The overall results suggest that Survivin, Bax and Bcl‐2 proteins may cooperatively contribute to cell apoptosis and cell proliferation in the cat uterus during the oestrus cycle.     

Morphological study of metaphase-specific apoptosis in MRL mouse testis

Apoptosis of male germ cells is a complex phenomenon in many animal species. Understanding its mechanisms could be useful in the diagnosis and therapy of male infertility. To examine the differences of distribution of apoptosis among mouse strains, the terminal transferase-mediated nick end labelling (TUNEL) method was employed. In the testes of MRL mice, many TUNEL-positive cells were identified at the metaphases of meiotic spermatocytes. Morphometrical analysis revealed that metaphase-specific apoptosis occurred at the region between secondary spermatocytes and step 1 spermatids in stage XII seminiferous tubules. In the investigation of the developing first-wave of seminiferous tubules, there were some metaphases showing apoptotic morphology prior to becoming secondary spermatocytes. Details of the apoptotic structure revealed by electron microscopy showed that cellular arrest occurred after the beginning of the M phase of the cell cycle. These results suggested that metaphase-specific apoptosis in the testis of the MRL mouse strain took place at least at the first meiotic division, perhaps showing the spindle assembly checkpoint of the cell cycle.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Immunohistochemical detection of polyunsaturated fatty acid oxidation markers in acetaminophen-induced liver injury in rats

To clarfity whether polyunsaturated fatty acid (PUFA) oxidation is involved in the mechanism of acetaminophen (APAP)-induced apoptotic cell death, the production and localization of PUFA oxidation markers N(ε)-propanoyl-modified lysine, N(ε)-hexanoyl-modified lysine, 4-hydroxyhexenal-modified histidine and crotonaldehyde-modified lysine were evaluated in the development of APAP-induced liver injury. The immunoexpression of these markers in the liver was examined up to 24 hr post-APAP intraperitoneal injection in rats (1 g/kg body weight). The histopathological changes in the liver appeared 3 hr after APAP injection and became exacerbated with time. Proapoptotic protein Bax immunoreactivity was first detected in the degenerative hepatocytes 3 hr after the injection and areas positively immunostained for Bax reached a peak level at 6 hr, and then decreased at 12 and 24 hr. There was a significant increase in the TUNEL-positive rate at 12 and 24 hr. Immunohistological expression of all these oxidation markers was first detected in the degenerative hepatocytes 3 hr after the injection, and earlier than the occurrence of hepatocyte apoptosis. Immunohistochemical expression of these markers were observed in almost all degenerative hepatocytes 3-24 hr after APAP injection. Areas positively immunostained for these markers reached a peak level at 6 hr, and then decreased at 12 and 24 hr. The results thus suggest that the generation of PUFA oxidation markers may be the signature of early events preceding the induction of liver cell apoptosis and thus useful for early detection of oxidative stress-related liver cell injury.     

Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor-I in feline fibroadenomatous change

The immunohistochemical expression, tissue-specific and cell-specific distribution patterns of progesterone receptors (PR), growth hormone (GH) and insulin growth factor-I (IGF-I) have been studied in 22 cases of feline fibroadenomatous change (FFAC). PR and GH were detected in all cases and were distributed homogeneously throughout the lesion, while IGF-I was detected in 77% of the cases at the site of ductal budding. The simultaneous expression of PR, GH and IGF-I was detected in epithelial cells in 14 of 22 cases while PR and GH expression only was detected in epithelial cells in 11 cases. Cases that expressed GH and IGF-I without PR expression in the stroma were the most numerous. Double immunohistochemical staining showed the co-localisation of PR and GH in a subset of ductal epithelial cells located between basal/myoepithelial and luminal cells (probably undifferentiated stem cells). These results suggest that ligand-activated progesterone receptors may induce the local synthesis of GH which in turn may exert its proliferative action directly and also indirectly through the production of other growth factors, such as IGF-I, in an autocrine/paracrine manner.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.