Proliferating Cell Nuclear Antigen Immunohistochemistry Research Articles

Anti-Müllerian hormone-mediated preantral follicle atresia is a key determinant of antral follicle count in mice.

Does anti-Müllerian hormone (AMH) induce preantral follicle atresia in mice? The present findings suggest that AMH-mediated follicle atresia only occurs in early follicles before they become sensitive to FSH. Most prior studies have investigated the ability of AMH to inhibit primordial follicle activation. Our previous study showed that AMH-overexpressing mice had fewer preantral follicles than expected after accounting for primordial follicle inhibition but the reason for this was not determined. Cross-sectional-control versus transgenic/knockout mouse studies were carried out. Studies were conducted on female wild-type (Amh+/+), AMH-knockout (Amh-/-) and AMH overexpressing (Thy1.2-AMHTg/0) mice on a C57Bl/6J background (age: 42-120 days). The follicle counts were conducted for primordial, transitioning, primary, secondary and antral follicles in Amh-/- and Amh+/+ mice. After confirming that follicle development speeds were identical (proliferating cell nuclear antigen immunohistochemistry), the ratio of follicles surviving beyond each stage of folliculogenesis was determined in both genotypes. Evidence for increased rates of preantral follicle atresia was assessed by active caspase-3 immunohistochemistry in wild-type and Thy1.2-AMHTg/0 mice. Amh -/- mice at 100-120 days of age had lower primordial follicle counts but higher primordial follicle activation rates compared to Amh+/+ mice. These counteracting effects led to equivalent numbers of primordial follicles transitioning to the primary stage in Amh+/+ and Amh-/- mice. Despite this, Amh+/+ mice had fewer primary, secondary, small antral and medium antral follicles than Amh-/- mice indicating differing rates of developing follicle atresia between genotypes. Cleaved caspase-3 immunohistochemistry in Thy1.2-AMHTg/0 ovaries revealed high rates of granulosa cell and oocyte apoptosis in late primary/early secondary follicles of Thy1.2-AMHTg/0 mice. N/A. The findings were shown only in one species and additional research will be required to determine generalizability to other species. This study is consistent with prior studies showing that Amh-/- mice have increased primordial follicle activation but these new findings demonstrate that AMH-mediated preantral follicle atresia is a predominant cause of the increased small antral follicle counts in Amh-/- mice. This suggests that the role of AMH is not to conserve the ovarian reserve to prolong fertility, but instead to prevent the antral follicle pool from becoming too large. While this study may demonstrate a new function for AMH, the biological purpose of this function requires further investigation, particularly in mono-ovulatory species. This study was funded by the Health Research Council of New Zealand and the University of Otago. No competing interests to declare.

Monitoring Cellular Proliferation and Apoptosis in Atherosclerosis Plaques and Intimal Thickenings.

Immunohistochemistry for specific proteins characteristic of proliferative or apoptotic cells allows for monitoring of these cell behaviors in biological tissues samples, including atherosclerotic plaques and intimal thickenings. Proliferating cell nuclear antigen (PCNA) and Ki-67 are widely used markers of cell proliferation and cleaved caspase-3 is a well-established marker of apoptosis that can be detected in tissue samples using immunohistochemistry. This technique enables quantification of the abundance of these proteins and provides information on the distribution of these biomarkers in tissues. By combining with immunohistochemistry for specific cell type markers, it is also possible to determine which cell types are proliferating or undergoing apoptosis. Here, we detail protocols for immunohistochemistry of PCNA, Ki-67, and cleaved caspase-3 for evaluation of cellular proliferation and apoptosis in atherosclerotic plaques in vivo. In addition, we outline methods for the quantification and localization of cell proliferation using bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) and ethynyldeoxyuridine/5-ethynyl-2 ́-deoxyuridine(EdU) labeled tissue samples collected from animals exposed to BrdU or EdU.

The Effect of Vanadium Inhalation on the Tumor Progression of Urethane-Induced Lung Adenomas in a Mice Model

Lung cancer has the highest death rates. Aerosol drug delivery has been used for other lung diseases. The use of inhaled vanadium (V) as an option for lung cancer treatment is explored. Four groups of mice were studied: (1) Saline inhalation alone, (2) Single intraperitoneal (i.p.) dose of urethane, (3) V nebulization twice a week (Wk) for 8 Wk, and (4) A single dose of urethane and V nebulization for 8 Wk. Mice were sacrificed at the end of the experiment. Number and size of tumors, PCNA (proliferating cell nuclear antigen) and TUNEL (terminal deoxynucleotidyl tranferase dUTP nick-end labeling) immunohistochemistry were evaluated and compared within groups. Results: The size and number of tumors decreased in mice exposed to V-urethane and the TUNEL increased in this group; differences in the PCNA were not observed. Conclusions: Aerosol V delivery increased apoptosis and possibly the growth arrest of the tumors with no respiratory clinical changes in the mice.

Reproductive aging and menopause-like transition in the menstruating spiny mouse (Acomys cahirinus).

Does the naturally menstruating spiny mouse go through menopause? Our study is the first to show a natural and gradual menopausal transition in a rodent. Age-related depletion of the human ovarian reserve (OvR) leads to menopause, the permanent cessation of menstruation and reproduction. Current rodent models of menopause are inappropriate for inferences of the human condition, as reproductive senescence is abrupt or induced through ovariectomy. The spiny mouse is the only confirmed rodent with a naturally occurring menstrual cycle. Histological assessment of virgin spiny mice occurred in females aged 6 months (n = 14), 1 year (n = 7), 2 years (n = 13), 3 years (n = 9) and 4 years (n = 9). Endocrinology was assessed in a further 9 females per age group. Five animals per group were used for ovarian stereology with additional ovaries collected at prenatal Day 35 (n = 3), day of birth (n = 5), postnatal Days 35 (n = 5) and 100 (n = 5) and 15 months (n = 5). Morphological changes in the reproductive system were examined using hematoxylin and eosin stains. Proliferating cell nuclear antigen immunohistochemistry assessed endometrial proliferation and sex steroids estradiol and testosterone were assayed using commercial ELISA kits. The proportion of females actively cycling was 86% at 6 months, 71% at 1 year, 69% at 2 years, 56% at 3 years and 44% at 4 years. Uterine and ovarian weights declined steadily from 1 year in all groups and corresponded with loss of uterine proliferation (P < 0.01). Estradiol was significantly decreased at 1 and 2 years compared to 6-month-old females, before becoming erratic at 3 and 4 years, with no changes in testosterone across any age. Fully formed primordial follicles were observed in prenatal ovaries. Aging impacted on both OvR and growing follicle numbers (P < 0.001-0.0001). After the age of 3 years, the follicle decline rate increased more than 5-fold. N/A. This is a descriptive study in a novel research rodent whereby reagents validated for use in the spiny mouse were limited. The gradual, rather than sudden, menopausal transition suggests that the spiny mouse is a more appropriate perimenopausal model than the current rodent models in which to examine the neuroendocrine pathways that encompass all hormonal interactions in the hypothalamic-pituitary-gonadal axis. The logistic, ethical and economic advantages of such a model may reduce our reliance on primates in menopause research and enable more thorough and invasive investigation than is possible in humans. Hudson Institute is supported by the Victorian State Government Operational Infrastructure Scheme. The authors declare no competing interests.

The Advantage of the Supercooling Storage Method for Transplantable Sources: Human Umbilical Vessel Endothelial Cells and Mouse Skin Grafts

The safety and efficacy of preserving transplantable tissue depends on multiple factors including temperature, length of preservation, and types of solvent. Supercooling storage, in which the preservation temperature goes below the freezing point without actual freezing of the tissue, has the potential to substantially extend the preservation time of cells, tissues, and organs. Herein we studied the effect of supercooling storage on preserving the viability of transplantable biomaterials. Human umbilical vein endothelial cells (HUVECs) and mouse dorsal skin grafts were stored at 2 different temperature (4°C and -4°C). The viability of these tissues was assessed using trypan blue exclusion assay, tetrazolium salt (WST-8) assay, and proliferating cell nuclear antigen immunohistochemistry analysis at various time points. Over time, the viability of HUVECs and mouse skin grafts decreased in each group and at both storage temperatures. The viability of HUVECs, evaluated with trypan blue exclusion assay and WST-8 assay, was better preserved during supercooled storage (-4°C) compared with refrigerated storage (4°C). Mouse skin grafts preserved under supercooled conditions showed less damage and a higher level of proliferating cell nuclear antigen expression. Among various preservation techniques, supercooling storage is 1 option to maintain optimal conditions for an increased organ transplantation success rate. To maximize preservation effectiveness, further investigations into the optimal supercooling temperatures, storage solvents, and cell protectants for various cells, tissues, and organs are needed.

Construction and Evaluation of Folic Acid-Modified 3-Bromopyruvate Cubosomes.

BackgroundDirect 3-bromopyruvate chemotherapy often causes side effects. We thus aimed to construct and evaluate folic acid-modified 3-bromopyruvate liquid crystalline nanoparticles (3BP-LCNP-FA) and assess their targeted antitumor effects in tumor-bearing nude mice.Material/MethodsA liquid crystalline nanoparticle formulation was screened, and the structure was characterized using polarizing light- and transmission electron microscopy. The folate target was then synthesized and characterized using differential scanning calorimetry and proton nuclear magnetic resonance spectroscopy. In vitro, human CNE-2Z and MDA-MB-231 tumor cells were used to evaluate 3BP-LCNP-FA effects on tumor cell morphology and proliferation. Different drug formulations were administered to tumor-bearing nude mice to observe the treatment effects. Hepatic and renal toxicities were assessed using hematoxylin and eosin-stained liver, kidney, and lung sections along with serological analysis of liver and kidney injury markers (e.g., aspartate aminotransferase, alanine transaminase, blood urea nitrogen, and creatinine). Tumor tissue was observed for changes using proliferating cell nuclear antigen immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay.ResultsWe successfully prepared 3BP-LCNP-FA of spherical shape with uniform size using the aforementioned techniques; drug loading did not alter crystal morphology. These cubosomes exhibited more potent antitumor activity than 3-bromopyruvate alone or non-folic acid-conjugated 3-bromopyruvate liquid crystalline nanoparticles in vitro and in vivo without obvious toxic side effects.ConclusionsIt is possible to successfully construct 3BP-LCNP-FA as a drug delivery vehicle that is more efficacious than 3-bromopyruvate and has no obvious toxic effects. Thus, folic acid-modified cubosomes can be used as effective carriers for targeted drug administration.

Histone deacetylase 4 deletion results in abnormal chondrocyte hypertrophy and premature ossification from collagen type 2α1‑expressing cells.

Histone deacetylase 4 (HDAC4) plays a vital role in chondrocyte hypertrophy and bone formation. To investigate the function of HDAC4 in postnatal skeletal development, the present study developed lineage‑specific HDAC4‑knockout mice [collagen type 2α1 (Col2α1)‑Cre, HDAC4d/d mice] by crossing transgenic mice expressing Cre recombinase. Thus, a specific ablation of HDAC4 was performed in Col2α1‑expressing mice cells. The knee joints of HDAC4fl/fl and Col2α1‑Cre, HDAC4d/d mice were analyzed at postnatal day (P)2‑P21 using an invivo bromodeoxyuridine (BrdU) assay, and Safranin O, Von Kossa and whole‑body staining were used to evaluate the developmental growth plate, hypertrophic differentiation, mineralization and skeletal mineralization patterns. The trabecular bone was analyzed using microcomputed tomography. The expressions of BrdU, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)‑13, runt‑related transcription factor (Runx)‑2, osteoprotegerin (OPG), CD34, type X collagen (ColX), osteocalcin and Wnt5a were determined using immunohistochemistry, in situ hybridization (ISH) and reverse transcription‑quantitative (RT‑q)PCR. The results demonstrated that HDAC4‑null mice (HDAC4d/d mice) were severely runted; these mice had a shortened hypertrophic zone (histopathological evaluation), accelerated vascular invasion and articular mineralization (Von Kossa staining), elevated expressions of MMP‑13, Runx2, OPG and CD34 (RT‑qPCR and immunohistochemistry), downregulated expression of the proliferative marker BrdU and PCNA (immunohistochemistry), increased expression of ColX and decreased expression of Wnt5a (ISH). In conclusion, chondrocyte‑derived HDAC4 was responsible for regulating chondrocyte proliferation and differentiation as well as endochondral bone formation.

Topical application of lectin Artin M improves wound healing in defects created in the palatal mucosa: an in vivo study in dogs.

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.

Effects of telmisartan and losartan on irradiated testes

AimsTo analyze the effects of radiation on the reproductive tissue of male Wistar rats and to evaluate whether treatment with the Ang II AT1 receptor antagonists telmisartan and losartan mitigate the dysfunctions resulting from this exposure. Main methodsRats were randomly divided into groups: Control, Irradiated, Telmisartan, Losartan, Irradiated+Telmisartan, and Irradiated+Losartan. Single dose of 5Gy was administered directly into the scrotum, followed by treatment with telmisartan (12mg/kg/day) or losartan (34mg/kg/two times/day) for 60days. Testicular function parameters were evaluated from spermatozoa of the vas deferens. Testes were processed for histopathological and morphometric-stereological analysis. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was evaluated. Key findingsRadiation significantly reduced sperm motility, concentration, vitality, and increased the number of abnormal spermatozoa. Telmisartan and losartan did not significantly prevent these radiation-induced disorders. Seminiferous tubules were atrophied in both untreated and treated irradiated testes, and exhibited vacuoles, increased interstitial tissue and high number of blood vessels. However, several seminiferous tubules in recuperation were founded among damaged tubules in the testes of treated animals. The PCNA immunohistochemistry confirmed these outcomes. PCNA-positive cells were detected in dividing spermatogonia and spermatocytes from irradiated telmisartan and losartan treated rats whereas in the only-irradiated group, PCNA staining was observed in the nuclei of only the surviving spermatogonia. SignificanceUnder these experimental conditions, the testicular function parameters showed that radiation produced marked damage that was not reversed by treatments. However, gonadal restructuring and recovery of spermatogenesis in treated animals may to reflect attenuation of radiation-induced damages and potential start of recovery.

Porcine Esophageal Submucosal Gland Culture Model Shows Capacity for Proliferation and Differentiation.

Background & AimsAlthough cells comprising esophageal submucosal glands (ESMGs) represent a potential progenitor cell niche, new models are needed to understand their capacity to proliferate and differentiate. By histologic appearance, ESMGs have been associated with both overlying normal squamous epithelium and columnar epithelium. Our aim was to assess ESMG proliferation and differentiation in a 3-dimensional culture model.MethodsWe evaluated proliferation in human ESMGs from normal and diseased tissue by proliferating cell nuclear antigen immunohistochemistry. Next, we compared 5-ethynyl-2′-deoxyuridine labeling in porcine ESMGs in vivo before and after esophageal injury with a novel in vitro porcine organoid ESMG model. Microarray analysis of ESMGs in culture was compared with squamous epithelium and fresh ESMGs.ResultsMarked proliferation was observed in human ESMGs of diseased tissue. This activated ESMG state was recapitulated after esophageal injury in an in vivo porcine model, ESMGs assumed a ductal appearance with increased proliferation compared with control. Isolated and cultured porcine ESMGs produced buds with actively cycling cells and passaged to form epidermal growth factor–dependent spheroids. These spheroids were highly proliferative and were passaged multiple times. Two phenotypes of spheroids were identified: solid squamous (P63+) and hollow/ductal (cytokeratin 7+). Microarray analysis showed spheroids to be distinct from parent ESMGs and enriched for columnar transcripts.ConclusionsOur results suggest that the activated ESMG state, seen in both human disease and our porcine model, may provide a source of cells to repopulate damaged epithelium in a normal manner (squamous) or abnormally (columnar epithelium). This culture model will allow the evaluation of factors that drive ESMGs in the regeneration of injured epithelium. The raw microarray data have been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus (accession number: GSE100543).

High temperature causes masculinization of genetically female olive flounder (Paralichthys olivaceus) accompanied by primordial germ cell proliferation detention

Sex determination is not only resort to a single genetic cascade but also regulated along a continuum of environmental and heritable factors in many fish species. Temperature is one of the most important non-genetic sex differentiation elements. Previous studies regarding high temperature masculinization of flatfish typically focused on sex ratios and reproductive endocrinology, but little was known about germ cells during the process. In this study, we investigated the dynamic changes of germ cells in terms of morphology and number employing serial-sectioning and the molecular investigation of expression of dnd and amh genes by qRT-PCR for qualitative and quantitative analysis of germ cell proliferation during sexual differentiation. Moreover, the proliferation activity of primordial germ cells was investigated using PCNA (proliferating cell nuclear antigen) immunohistochemistry.Experimental results revealed that high temperatures caused juvenile olive flounder masculinization (male ratio, 95.24%, 27.5°C±0.5°C) in comparison with controls (male ratio 5.56%, 18°C±0.5°C). The proliferation pattern of germ cells presented sexual dimorphism, from 42 to 66days post-hatching (dph). The experimental group presented specifically slow proliferation and greatly reduced cell numbers. No cyst clusters were apparent in comparison to the control group. Consistently, the proliferation activity of germ cells in the experimental group was significantly reduced in comparison to that observed in the control group at 50dph by PCNA immunohistochemistry. Furthermore, the expression levels of poamh during the process of temperature-induced masculinization gradually increased and were significantly higher than that in the control group at the stage gonads have been considered still undifferentiated in particular from a histological point of view. Thus, elevated temperatures result in masculinization, poamh up-regulation, accompanied by germ cell detention proliferation.

Disruption of Estrogen Receptor Alpha in Rats Results in Faster Initiation of Compensatory Regeneration Despite Higher Liver Injury After Carbon Tetrachloride Treatment.

Estrogen receptor alpha (ESR1) is 1 of the 2 intracellular receptors for estrogen and is expressed by hepatocytes in the liver. The role of ESR1 in the regulation of toxicant-induced liver injury and compensatory regeneration is not completely clear. We investigated the role of ESR1 in liver regeneration after carbon tetrachloride (CCl4)-induced liver injury using wild type (WT) and ESR1 knockout (ESR1-KO) rats. Adult female WT and ESR1-KO rats were treated with 1 mL/kg CCl4 and euthanized over a time course of 0 to 48 hours. Liver injury measured by serum alanine amino transaminase, and histopathological analysis showed significantly higher liver injury in ESR1-KO as compared to WT rats. Hematoxylin and eosin staining revealed 2-fold higher necrosis and significant inflammatory cell infiltration in ESR1-KO rats. Chloracetate esterase staining revealed higher neutrophil infiltration in ESR1-KO rat livers. Interestingly, proliferating cell nuclear antigen immunohistochemistry showed that in spite of 2-fold higher liver injury, the ESR1-KO rats had equal liver regeneration as compared to WT rats. Western blot analysis of cyclin D1 and phosphorylated Rb, proteins involved in the initiation of the cell cycle, was significantly higher at all time points in ESR1-KO rats. Further analysis revealed faster activation of canonical Wnt/β-catenin and NF-κB signaling in ESR1-KO rats characterized by higher activated β-catenin and phosphorylated p65 at 12 hours after CCl4 treatment. Taken together, these data indicate that ESR1-mediated signaling inhibits liver regeneration by downregulation of Wnt signaling resulting in lower cyclin D1 activation after chemical-induced liver injury.

Follicular development and morphological changes in the vaginal epithelium during the estrous cycle of Galea spixii.

The current study aimed to determine if characteristics observed in vaginal cytology during the estrous cycle of female SYT cavies corresponded with proliferation of the vaginal epithelium, characterized by proliferating cell nuclear antigen (PCNA) immunolocalization, and with follicular development at different phases of the estrous cycle. After determining estrous cycle phases by vaginal cytology, females were euthanized at metestrus, diestrus, proestrus, and estrus. Histological study of the vaginal epithelium and ovary were then performed. Immunohistochemistry for PCNA in vaginal tissue at each cycle phase was also performed. Superficial cornified cells and early post-ovulatory follicles were found at estrus. Few nuclei below the enucleate superficial cells were immunoreactive to PCNA. At metestrus, the vaginal epithelium underwent desquamation and lost the superficial cornified cells; basal and intermediate cells appeared, and the post-ovulatory follicle formed an early corpus luteum. No PCNA immunoreactivity was observed. At diestrus, the corpus luteum was developed, and the vaginal epithelium contained basal and intermediate cells. There was PCNA immunoreactivity in the cellular nucleus in the germinative stratum of the epithelium. Because of the growth and maturation of ovarian follicles, the vaginal epithelium suffered intense proliferation at proestrus. Vaginal cytology revealed large intermediate cells and nucleated and enucleated superficial cornified cells. In the ovary, mature follicles were present. More apparent immunoreactivity of PCNA in the germinative layer was found. In summary, we inferred that vaginal exfoliative findings matched the proliferation process of the vaginal epithelium. PCNA immunolocalization occurred as well as corresponding follicular development in the ovaries.

Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts.

To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon's capsule, both in vitro and in vivo. Human primary Tenon's capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT) method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1,2) and proliferating cell nuclear antigen (PCNA). Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d). A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson's trichrome to compare the neovascularization in the subconjunctiva area. In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3(rd), 7(th), 14(th) and 21(st) days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28(th) day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson's trichrome observation. By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

Characterization of mammary adenocarcinomas in male rats after N-methyl-N-nitrosourea exposure—Potential for human male breast cancer model

The frequency of breast cancer in men is extremely rare, reported to be less than 1% and there is currently no available animal model for male mammary tumors. We compared the characteristics of various immunohistochemical markers in N-methyl-N-nitrosourea (MNU)-induced mammary adenocarcinomas in male and female Crj:CD(SD)IGS rats including: estrogen receptor α (ER), progesterone receptor (PgR), androgen receptor (AR), receptor tyrosine-protein kinase erbB-2 (HER2), GATA binding protein 3 (GATA3), and proliferating cell nuclear antigen (PCNA). Female mammary adenocarcinomas were strongly positive in the nuclei of tumor cells for PCNA and ER (100%) with only 60% and 53% expressing PgR and GATA3, respectively. 100% of male adenocarcinomas also exhibited strongly positive expression in the nuclei of tumor cells for PCNA, with 25% expressing AR and only 8% showing positivity for ER. Male carcinomas did not express PgR or GATA3 and none of the tumors, male or female, were positive for HER2. Based on the observed ER and PgR positivity and HER2 negativity within these tumors, MNU-induced mammary adenocarcinomas in female rats appear to be hormonally dependent, similar to human luminal A type breast cancer. In contrast, MNU-induced mammary adenocarcinomas in male rats showed no reactivity for ER, PgR, HER2 or GATA3, suggesting no hormonal dependency. Both male and female adenocarcinomas showed high proliferating activity by PCNA immunohistochemistry. Based on our literature review, human male breast cancers are mainly dependent on ER and/or PgR, therefore the biological pathogenesis of MNU-induced male mammary cancer in rats may differ from that of male breast cancer in humans.

Expression pattern of proliferating cell nuclear antigen (PCNA) in round cell tumors of dogs from Grenada, West Indies

A retrospective study was performed on archivedtumor tissues received by the Veterinary Pathology Diagnostic Laboratory, School of Veterinary Medicine, St. George's University, Grenada over an 11 year period (2001–2011). The expression pattern of proliferating cell nuclear antigen (PCNA) was evaluated in canine round cell tumors. A total of 40 tumors comprising of transmissible venereal tumor(TVT), canine cutaneous histiocytoma(CCH)and mast cell tumor(MCT) were evaluated for PCNA expression using immunohistochemistry technique. Moderate to strong immunoreactivity was detected in most of these tumors. The percentage of immunomarked cells for PCNA was 42.1, 57.1, and 57.1 for TVT, CCH, and MCT, respectively. A statistical significance (P<0.05) between the average mitotic counts and PCNA expression for TVT and CCH was noted. PCNA expression in the TVT cases ranged from 17.4% to 56.3% which suggests the presence of variable growth fraction in this tumor. The results showed that PCNA immunohistochemistry could be an important adjunct tool in determining the biological behavior of round cell tumours.

Avaliação histomorfométrica e da proliferação celular em úlceras de córnea superficiais, induzidas em coelhos, após o uso de colírio de Citrus lemon

O objetivo do presente estudo foi avaliar e comparar, por meio de histomorfometria e imuno-histoquímica para PCNA (Antígeno Nuclear de Proliferação Celular), o processo de reparação corneal de úlceras superficiais induzidas em coelhos e tratadas com colírios de óleo essencial de Citrus lemon (CL). Foram utilizadas 40 fêmeas da espécie leporina, constituindo-se quatro grupos experimentais de 10 animais cada. Todos os animais foram submetidos à indução da úlcera superficial experimental por meio da aplicação tópica de n-heptanol. Em dois grupos foram instilados colírios à base de óleo essencial de Citrus lemon, em diferentes concentrações, sendo 3% (GL3) e 5% (GL5). Outro grupo foi tratado com Tween 80 8% (GT), que é o diluente utilizado na produção dos colírios de CL; o grupo controle (GC) recebeu apenas substituto da lágrima. Todos os colírios foram aplicados quatro vezes ao dia. Os grupos experimentais foram distribuídos em dois subgrupos, com cinco animais cada, de acordo com os períodos finais de avaliação. O primeiro subgrupo (M1) foi avaliado após 24 horas e o segundo (M2), após cinco dias. Nas comparações entre os momentos iniciais e finais, os grupos tratados com substituto da lágrima, Tween 80 8% e colírio à base de óleo essencial deCitrus lemon 5% promoveram aumento na espessura epitelial na periferia da córnea e maior percentual de proliferação celular. Não houve diferença de celularidade entre os tratamentos. Os colírios à base de óleo essencial de Citrus lemon, nas diferentes concentrações, promoveram a reepitelização corneal, sem causar lesões adicionais ao epitélio ou estroma corneal, podendo ser utilizado na superfície ocular.

PCNA and apoptosis during post-spawning ovarian remodeling in the teleost Oreochromis niloticus.

The balance between cell proliferation and apoptosis is crucial for tissue development and homeostasis. The present study investigated the contribution of proliferating cell nuclear antigen (PCNA) and apoptosis during ovarian remodeling after spawning in the Nile tilapia Oreochromis niloticus. Breeding females were kept in controlled conditions and ovary samples were collected weekly for TUNEL assay, immunohistochemistry for PCNA and caspase-3 and morphometric analysis. During the follicular growth, PCNA labeled mainly the nuclei of oocytes and follicular cells in a high proportion of follicles especially in primary growth, while a low occurrence of apoptosis in follicular and theca cells was detected. At 0-3 days post-spawning, post-ovulatory follicles showed no proliferative activity, however the follicular cells exhibited high rates of apoptosis. At 7-10 days, PCNA labeled the thecal cells in a low proportion of post-ovulatory follicles, which showed follicular cells with lower rates of apoptosis. PCNA labeled mainly the theca in the advanced and late stages of atretic follicles, while the follicular cells exhibited a significant increase of apoptosis along follicular atresia. We concluded that PCNA and apoptosis work cooperatively to ensuring the success of follicle development and maintaining of tissue homeostasis during follicular growth. PCNA and apoptosis are also essential mechanisms in the follicular regression during post-spawning ovarian recovery in the Nile tilapia.

N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorders.

Determination of cell proliferative activity by immnunohistochemical detection of proliferating cell nuclear antigen in N‐methyl nitrosoureainduced rat mammary tumours

Rodent experimental models are considered useful experimental systems to study mammary cancer, which occurs in rodents as a result of hormonal imbalance, much like the breast cancer in women. Rodent cells are easier to transform by chemical carcinogen due to their relatively poorer controls of genetic stability and DNA repair systems. Proliferating cell nuclear antigen (PCNA) is one of the most sensitive biomarkers to measure the cell proliferative activities in the neoplasms, especially when the otherwise undetectable PCNA protein over‐expresses to the immunohistochemically detectable levels. In the present study, mammary tumours were induced in Sprague Dawley rats by intraperitoneal administration of N‐methyl nitrosourea (MNU) chemical carcinogen. Palpable tumours were noticed after 60 days postinjection (dpi) with average latency period of 144 days, and 12 (46.15%) of 26 rats developed mammary tumours. A total of 18 tumours were diagnosed in 12 rats, including 2 cases (10%) of hyperplasia, 1 (5%) benign and 17 malignant tumours (85%). Malignant tumours included in situ ductal papillary carcinoma (11.11%), in situ ductal cribriform carcinoma (38.88%), invasive tubular adenocarcinoma (5.56%), invasive papillary carcinoma (16.67%) and invasive cribriform carcinoma (22.22%) were. PCNA immunohistochemistry revealed moderate to strong nuclear staining in neoplastic cells, with highest mean PCNA index of 55.53±10.13 in invasive cribriform carcinoma. The PCNA indices were significantly different (p&lt;0.01; one way ANOVA) between control mammary gland, hyperplasia, benign and malignant tumours. It was concluded that the measurement of PCNA expressing cells employing immunohistochemistry can help in the determination of the level of malignancy in MNU‐induced rat mammary tumours.