Apoptosis in the canine endometrium during the estrous cycle

Apoptotic cell death in the endometria of 58 female dogs in different stages of the estrous cycle was assessed (in formalin-fixed, paraffin-embedded sections) with both the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and immunohistochemical detection of caspase-3 activity. For both techniques, the apoptotic index was determined in the surface epithelium, stroma, crypts, and basal glands by counting the percentage of stained cells in a total of 500 cells in each category. In the surface epithelium and stroma, TUNEL- and caspase-3-positive cells were rare (apoptotic index<1) throughout the estrous cycle. However, caspase-3 detection showed a significant increase in the apoptotic index in the stroma during anestrus as well as an increase in the index in both the stroma and surface epithelium in late metestrus. The apoptotic index increased during late metestrus and anestrus in the crypts and basal glands; in the crypts, this increase was significant only when caspase-3 detection was used, whereas in basal glands, significant differences were found for both techniques. In conclusion, apoptosis was present in canine endometrial cells during the estrous cycle, but caspase-3 detection showed more significant differences than the TUNEL assay. Furthermore, a high apoptotic index (suggestive of endometrial desquamation) was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and serum progesterone concentrations.     

Spatial distribution and cell kinetics of the glands in the human esophageal mucosa

The glands in the human esophageal mucosa have been considered rudimentary, and have been poorly studied. Only recently, their role in the defense of the esophageal mucosa in gastroesophageal reflux disease has been put in evidence. In the present study, the presence of the different esophageal gland types was observed in 82 necropsy specimens. A precise topographical study was possible in 15 specimens. Cell proliferation parameters, namely DNA synthesis and mitotic index values in the squamous epithelium and in the esophageal glands were measured in 8 patients undergoing a surgical palliative esophagectomy, after in vivo labelling with bromodeoxyuridine. Upper mucosal, lower mucosal, and submucosal glands were observed in respectively 3.7%, 87% and 99% of the 82 esophageal specimens. Our topographical analysis showed that the upper mucosal glands, lower mucosal glands, and submucosal glands are occupying respectively 0.02%, 2.61%, and 3.96% of the esophageal surface. DNA synthesis and mitotic index values in the progenitor zone of the squamous epithelium were respectively 7.91% and 0.81%, whereas the proliferative activity in the glands was extremely low. The labelling index In the excretory ducts of the glands was only 0.07%, while no labelled cells nor mitotic figures were observed in any of the glandular acini.     

Detection of estrogen alpha and progesterone receptors and cell proliferation in the uterus during early pregnancy in the goat

The objectives of this study were to investigate in the goat uterus the expression of estrogen-alpha (ER alpha) and progesterone receptors (PR) and their relationship to proliferation indices (Ki-67) during peri-implantation on Days 22 to 30 post coitum (pc). Immunohistochemical methods were used to quantify ER alpha and PR for luminal and deep regions of the endometrium and of the myometrium. On Day 22 pc cell proliferation was only observed in the luminal epithelium. On Day 24 pc, high cell proliferation indices were seen in luminal epithelium and proliferation began in the luminal stroma and glands. There was a positive correlation between Ki-67 and total ER alpha score in the luminal epithelium (r = 0.53, P < 0.01). Levels of PR scores were highly correlated with Ki-67 indices in luminal epithelium (r = 0.74, P < 0.01) and stroma (r = 0.70, P < 0.01). No Ki-67 expression was observed in deep glands, stroma or myometrium on any of the days studied. Results indicate that patterns of ER alpha and PR expression differ markedly, and that there was a high correlation between PR expression and cell proliferation in the caprine uterus during the peri-implantation period.     

Effects of selective oestrogen receptor modulators on proliferation in tissue cultures of pre- and postmenopausal human endometrium

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle, 17beta-estradiol (17beta-E2, 1nM), oestrogen receptor (ER) antagonist ICI 164.384 (40nM), and 4-OH-tamoxifen (40nM), raloxifene (4nM), lasofoxifene (4nM) and acolbifene (4nM). Protein expression of ERalpha, ERbeta1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17beta-E2 increased the fraction of Ki-67 positive cells (p<0.001) by 55% in glands compared to the control. Raloxifene (4nM) increased (p<0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p<0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium.     

Sex steroid receptor expression in the oviduct and uterus of sheep with estrus synchronized with progestagen or prostaglandin analogues

The objective of this study was to investigate differences in the expression of estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and the proliferative indexes (Ki-67), in the uterus and oviduct of sheep with estrus synchronized either by prostaglandin analogues (Group PA, n=27) or by treatment with progestagens (Group P, n=29) on days 4 and 7 (day 0=estrus), when the embryos were collected. Immunohistochemical methods were used to quantify ERalpha, PR and Ki-67 in six superficial and deep compartments in the uterus and oviduct. The expression of ERalpha was significantly (P<0.01) lower in progestagen treated ewes than in prostaglandin analogues treated group in the luminal epithelium, superficial glands and superficial stroma in the uterus on day 4. The expression of PR was significantly lower in progesterone treated ewes than in the PA Group in the superficial gland (P<0.05) in both days studied. The lowest expression of PR was observed in the luminal caruncular epithelium and superficial glands in both treatments, obtaining the lowest levels on day 4 (P<0.05). There were significant differences between days 4 and 7 in the Ki-67 immunostaining in the luminal epithelium (P<0.01) and superficial glands (P<0.05). A higher cell proliferation was observed in the uterine epithelium (P<0.05) on day 4 in the animals treated with progestagens. Results indicate that sheep with synchronization of estrus with progestagens showed a reduction of ERalpha and PR protein expression in most of oviductal and uterine cells.     

Combined Ki-67 and feulgen stain for morphometric determination of the Ki-67 labelling index

In this note we present a combined Ki-67 and Feulgen stain for morphometric determination of the Ki-67 labelling index. The immunohistochemical part of this double staining technique is based on the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method, visualizing the enzyme activity by the nitro-blue-tetrazolium chloride (NBT)/bromo-chloro-3-indolyl-phosphate (BCIP) technique. The NBT/BCIP complex resists the hydrolytic activity of the Feulgen stain. The staining method presented allows semi-automatic determination of both the total nucleus-area as well as the Ki-67 positive nucleus-area using a morphometric computer system. The Ki-67 labelling index thus achieved is based on the relative nuclear area of Ki-67 positive nuclei and is clearly more precise and efficient than the counting method using an ocular grid.     

Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.     

Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle.

Immunohistochemical detection of the proliferation marker Ki-67 antigen was used to monitor mitotic activity in the endometrium of mares. The monoclonal antibody MIB1 was validated for use on equine tissues by demonstrating its reaction with activated peripheral blood lymphocytes, and endometrial biopsies were recovered from 26 non-pregnant mares at selected stages during the reproductive cycle. The proportion of positively stained nuclei was counted in five random areas on each histological section to determine the percentage and type of proliferating cells. Multiplication rates in the types of cell found in the superficial strata, comprising the luminal epithelium, the epithelium of the gland necks and the stromal cells of the stratum compactum, were greatest during oestrus, presumably under the influence of oestrogens secreted by the growing ovarian follicles. In contrast, the mitotic activity in the cells of the deeper secretory portions of the endometrial glands was restricted to a brief phase between day 3 and day 7 of dioestrus, most likely as a delayed response to the decreasing oestrogen concentrations after ovulation. Some of the degenerate glands in subfertile mares did not follow this pattern of increased epithelial proliferation at that stage. After day 7 of dioestrus, the proliferation rates of cells in the endometrium decreased to basal values and remained low for as long as progesterone concentrations remained evaluated, even during prolonged dioestrus. The technique enabled characterization of normal cell proliferation patterns in the endometrium of mares and it will be a useful tool in the future for monitoring the endometrial responses of reproductively healthy and subfertile mares.     

Real-time quantification of the proliferative state in astrocytomas

OBJECTIVE: To evaluate proliferative activity in a set of gliomas and to compare the quantitative data obtained by a real-time processor with the labelling index (LI) and mitotic index (MI). STUDY DESIGN: Ki-67 immunostaining was performed on paraffin-embedded specimens from 42 cases of glioblastomas, 17 cases of anaplastic astrocytomas and 14 cases of low grade astrocytomas. Nuclear positivity was calculated as LI and by a real-time image processor for quantitative evaluation. MI was also calculated at 10 high-power fields. The data obtained from glioblastomas were compared with those from anaplastic and low grade astrocytomas. To all the data was applied the Pearson test to verify the correlation between counting and quantitative values and between proliferative markers and survival. RESULTS: A positive trend from low grade astrocytomas to glioblastomas was found for Ki-67 (LI and quantitative values) and MI, with highly significant differences between the three grades of gliomas considered. A good correlation between LI and quantitative values of Ki-67 was found. Very little relationship resulted between survival and Ki-67 LI. No relationship was found between survival and quantitative values of Ki-67. CONCLUSION: Ki-67 allowed effective separation of astrocytic tumors with different grades of malignancy. Quantitative evaluation of color information by means of a real-time processor proved to be a useful, objective and fast way to obtain readings, useful for grading purposes but not for prognostic evaluation.     

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.     

A comparison of different methods to determine cell proliferation by flow cytometry

Abstract. The proliferation of human melanoma cells (MeWo) in vitro was studied with a number of different techniques. In particular, we compared the expression of PCNA and the Ki-67 antigen on the one hand with BrdU pulse and continuous labelling on the other. Two-dimensional flow cytometry (with DNA content as a second parameter) was employed to discriminate between cycling and non-cycling cells as well as cells in the G₁, S and G₂ phases of the cycle. Cell cultures in different stages of growth were analyzed. We found that the percentage of anti-PCNA and Ki-67 positive cells agreed very well with the BrdU pulse and continuous labelling index, respectively. Our data further support the assumption that under certain conditions PCNA is a marker of S-phase cells, whereas Ki-67 can be used to quantify the growth fraction. Possible pitfalls of the techniques are discussed.     

The involvement of proliferation and apoptosis in the early human gonad development

Distributions of the Ki-67, TP53, caspase-3 and AIFM1 markers were histologically investigated in the 5th to 9th week developing gonads of 12 human conceptuses using immunohistochemical and immunofluorescence methods. Between the 5th and 8th developmental week, proliferation gradually increased in the surface gonad epithelium (26–52 %) and stroma (19–42 %), but then slightly decreased in the surface epithelium (35 %) during the early foetal period. In medulla, low proliferation activity decreased from 15 to 12 % between the 7th and 9th week. At earliest stages of gonadal development, primordial germ cells (PGC) were only rarely TP53 positive. In the 7th and 8th week, almost all PGC-s displayed TP53 positivity, while their number decreased in early fetal period. During the investigated period, caspase-3 reactivity gradually decreased in surface epithelium, while it increased in PGC and medulla of developing gonad AIFM1-positivity first appeared in surface gonad epithelium and then predominantly in PCG-s while caspase-3 characterized different cell populations within the developing gonad. AIFM1 and caspase-3 co-localized only during the migration of PCG-s. The number and distribution of Ki-67, TP53, caspase-3 and AIFM1 reacting cells changed coincidently with development end regression of the sex cords in indifferent and early fetal gonad. Our results indicate that the number of PGC might be controlled by balance of TP53 and AIFM1, leading to caspase-3 independent cell death. Other cell populations are probably eliminated by caspase-3-dependent cell death. Both pathways of cell death seem to operate during early human gonad development, while their intensity varies depending on the cell type and developmental period analysed.     

Intestinal epithelium in Anarhichas lupus L., with emphasis on cell renewal

Histomorphological studies on the dynamics of intestinal epithelium in larvae, juveniles and adult stages of common wolffish Anarhichas lupus showed that the major structural development took place during the first 8 weeks after hatching ( c . 320 day degrees). This development preceeded a period of increase in relative growth. The mucosa of the proximal and distal intestine gradually developed from simple primary mucosal folds into a complex structure of primary and secondary folds with regularly distributed crypts. Cell proliferation, based on the observations of the presence of immature cells, mitotic figures and proliferating cell nuclear antigen reacting cells, was limited to the crypts after 8 weeks of age. The observation of bands of epithelial cells showing apoptotic and necrotic changes, suggested the existence of extrusion zones, where effete cells were eliminated by both shedding and phagocytosis. In the proximal intestine of adult common wolffish, increasing amounts of lipids were observed from the upper part of the crypts to the surface epithelium, probably reflecting an increased maturation of epithelial cells.     

Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice

Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.     

Localization of NADPH-diaphorase activity in the dental pulp,periodontium and alveolar bone of the rat

In this study we examined the presence and localization of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the dental pulp, periodontal tissues and alveolar bone of the rat. The presence of NADPH-d activity was also examined in cat pulp. The rat histochemical analysis revealed the presence of prominent NADPH-d activity both in cells of the sub-odontoblastic cell layer and in the odontoblasts, in the root as well as in the coronal pulp regions. In the pulpal horns, odontoblasts often had long processes with a high level of labelling indicating NADPH-d activity extending through the predentin and dentin. Moreover, endothelial cells of pulpal blood vessels were positive for NADPH-d in both species. However, no clearcut examples were found of pulpal nerve fibres positive for NADPH-d in the rat or cat and denervation performed in rats did not alter the enzyme staining patterns. In the periodontal tissue, NADPH-d activity was localized to cells on the alveolar bone surface of the periodontal ligament and, in addition, alveolar bone marrow crypts were filled with intensely labelled cells. In the gingival papillae, NADPH-d activity was observed in the basal cell layer of the epithelium. Endothelial cells of periodontal and gingival blood vessels showing positive staining for NADPH-d were occasionally noted.     

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Expression of cyclin-dependent kinase inhibitors in taste buds of mouse and hamster

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.     

Ki-67 monoclonal antibody (MAb) reacts with a proliferation associated nuclear antigen in the rabbitOryctolagus cuniculus

Summary The Ki-67 monoclonal antibody which recognizes a human nuclear antigen expressed by cycling cells but not by resting cells was found to react immunohistochemically with tissues from the rabbitOryctolagus cuniculus. Ki-67 immunoreactivity was restricted to the nucleus. A comparative study with bromodeoxyuridine labelling patterns was carried out to study the association with proliferating cells. In lingual, jejunal and appendix mucosa, skin, adrenal gland, thymus, spleen, bone marrow, testis, growth cartilage, periosteum and perichondrium of long bones the distribution of Ki-67 positive and bromodeoxyuridine labelled cells was similar and consistent with the distribution of proliferating cells in these tissues. In tissue from the brain, kidney, skeletal or cardiac muscle and liver no Ki-67 positive or bromodeoxyuridine labelled cells were seen. In cartilage labelled in vivo with tritiated thymidine, all thymidine labelled cells were also Ki-67 positive. These results suggest that the Ki-67 antibody recognizes a nuclear antigen in the rabbit that is associated with cell proliferation and is expressed by cells in S-phase as well as in other phases of the cell cycle.     

Ki-67 monoclonal antibody (MAb) reacts with a proliferation associated nuclear antigen in the rabbit Oryctolagus cuniculus

The Ki-67 monoclonal antibody which recognizes a human nuclear antigen expressed by cycling cells but not by resting cells was found to react immunohistochemically with tissues from the rabbit Oryctolagus cuniculus. Ki-67 immunoreactivity was restricted to the nucleus. A comparative study with bromodeoxyuridine labelling patterns was carried out to study the association with proliferating cells. In lingual, jejunal and appendix mucosa, skin, adrenal gland, thymus, spleen, bone marrow, testis, growth cartilage, periosteum and periochondrium of long bones the distribution of Ki-67 positive and bromodeoxyuridine labelled cells was similar and consistent with the distribution of proliferating cells in these tissues. In tissue from the brain, kidney, skeletal or cardiac muscle and liver no Ki-67 positive or bromodeoxyuridine labelled cells were seen. In cartilage labelled in vivo with tritiated thymidine, all thymidine labelled cells were also Ki-67 positive. These results suggest that the Ki-67 antibody recognizes a nuclear antigen in the rabbit that is associated with cell proliferation and is expressed by cells in S-phase as well as in other phases of the cell cycle.     

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19( )positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro.