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This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKC1) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in fol- licular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein -protein interactions regu- lating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation exper- iments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER þ HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKC1. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKC1. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKC1 is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

Adenomatous polyposis coli (APC) is a tumor suppressor gene whose main function is the destabilization of beta-catenin, a key effector of the Wnt signaling pathway. This gene is defective in familial adenomatous polyposis (FAP), a dominantly inherited disease, but inactivation of APC has been reported also in most sporadic colorectal tumors and it is considered an early event in colorectal tumorigenesis. The aim of the present study was to evaluate the intracellular ultrastructural distribution of beta-catenin and APC proteins in epithelial cells of normal colorectal mucosa, aberrant crypt foci (ACF, an early premalignant lesion) and cancer. We used the immunogold electron microscopic method to identify both proteins. Normal colonic epithelial cells showed a strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Normal cells showed APC localization pattern characterized by diffuse nuclear expression and along the plasma membrane. In ACF and in carcinoma an absent or reduced membranous expression of beta-catenin was associated with an increased nuclear and cytoplasmatic expression. In aberrant crypt foci and carcinoma, APC was evident inside the nucleus and at the level of cell-cell junctions, but it was decreased in the cytoplasm. This method allowed the accurate localization of proteins of the Wnt signaling pathway in the early steps of colorectal carcinogenesis. The similar pattern of subcellular distribution of APC and beta-catenin in dysplastic ACF and colorectal cancer suggests that ACF are precursor lesions of sporadic and FAP-associated colorectal carcinoma.

Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene‐blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans.

During human oogenesis two contrasting processes can be observed: germ cell proliferation and differentiation, and contemporaneous germ cell death. It is well known that apoptosis is a type of physiological cell death that occurs in proliferating and differentiating tissues. The aim of this study is to demonstrate, through ultrastructural and in-situ 3' end labelling observations in intact sections of human fetal ovaries, that germ cell loss during fetal life is due to a phenomenon of apoptosis. We evaluated the presence of programmed cell death in female germ cells in fetal ovaries at 18-20 weeks of postconceptional age. The alterations that occur during apoptosis were detected by the electron microscope and include cytoplasmic condensation, organelle relocalization and compaction, chromatin condensation, and finally, production of membrane-enclosed particles containing intracellular material, known as apoptotic bodies, that are phagocytosed. The fragmentation of DNA, characteristic of apoptotic cells, was detected by the use of the in-situ 3' end labelling procedure on histological sections of ovaries where only some nuclei of germ cells were positively stained. The parallel use of these two methods on human ovaries of 18-20 weeks postconceptional age has allowed us to show that the numerical decrease of human female germ cells during the fetal period is due to an apoptotic phenomenon.

Matrix metalloproteinases (MMPs) have been well characterized for their ability to degrade extracellular matrix proteins and, thus, they have been studied to elucidate their involvement in both tumor development and progression. In the present study, attention was focused on MMP-15 and MMP-19, two less known members of the MMP family. The expression profile of MMP-15 and -19 was assayed in samples of normal colorectal mucosa, microadenomas and cancer using confocal analysis, western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both qRT-PCR and western blotting showed that MMP-15 and MMP-19 appeared to be upregulated during colorectal tumorigenesis, with different expression patterns: MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer with respect to microadenomas; the semiquantitative immunofluorescence analysis showed a stromal localization of this protein in the early phases of neoplastic transformation. Increasing amount of MMP-19 mRNA and protein levels were observed in the progression of colonic lesions; MMP-19 staining increased in the normal mucosa-microadenoma-carcinoma sequence. Such different expression patterns, are probably due to the different roles played in colorectal tumorigenesis by these two molecules. Conflicting data on the role of these proteins in tumor progression have been reported, thus, an improved understanding of the biological roles of MMPs, in particular the lesser known members such as MMP-15 and 19, in colorectal cancer may lead to a re-evaluation of the use of MMP inhibitors and suggests the need of integrated translational studies on MMP expression patterns.

Chronic mucosal inflammation is considered a risk factor for colorectal cancer. Neutrophils are a major source of oxidants, whereas cyclooxygenase 2 (COX-2) and Hypoxia Inducible Factor-1alpha (HIF-1alpha) protein expression levels are increased in inflammatory and malignant lesions. The main purpose of the present study was to evaluate myeloperoxidase (MPO) positive cell infiltration, COX-2 and HIF-1alpha protein expression in colorectal carcinogenesis, especially in its early phases, using immunohistochemistry and immunofluorescence confocal microscopy techniques. MPO, COX-2 and HIF-1alpha proteins were expressed at higher rates in the normal colorectal mucosa of patients with inflammatory bowel diseases and colorectal tumours than in patients with normal colonoscopy. A gradual increase in COX-2 and HIF-1alpha protein expression was observed in dysplastic aberrant crypt foci, adenomas and carcinomas, showing a strong relation to dysplasia. In conclusion, the present study supports the hypothesis of a key role of inflammation in malignant transformation of colorectal mucosa. The evaluation of some early markers related to inflammation in the mucosa of the large bowel may serve as potential tool for prognosis and therapeutic strategies.

Extracorporeal shockwave lithotripsy (SWL) is one of the most common treatments for urinary stones. Despite technological improvements, it may cause side effects varying from minor reversible microscopic damage to severe large renal hematomas. The aim of our experimental study is to assess the efficacy of inosine in avoidance of acute renal damage after SWL.We used 25 Wistar rats that had previously had left nephrectomy. The rats were divided into three groups: group A consisted of 10 rats undergoing renal SWL; group B consisted of 10 rats that received adjunctive treatment with IP injection of inosine 40 minutes before SWL; and group C consisted of 5 rats that served as controls. N-acetylglucosaminidase (NAG) and lactate dehydrogenase (LDH) concentrations were evaluated 24 hours before and 24 hours after SWL. All the rats were subsequently sacrificed (4 rats in group A and 4 in group B at 48 hours post-SWL, and the remaining rats were sacrificed 30 days post-SWL). Renal tissue was submitted to histologic and electron microscopic examination to assess early and late alterations.NAG and LDH values were significantly increased after SWL in group A (P0.001), while no significant NAG and LDH differences were detected in group B (P0.16). Early histologic examination revealed a considerable amount of cellular degeneration in group A with ultrastructural vacuolization and disruption of lysosomal membranes; the tubular features and cellular structures appeared to be well preserved in group B. No late histologic alterations were evident in any of the specimens.Inosine is helpful and protective in the prevention of early microscopic damage to renal parenchyma due to SWL.

A morphometric analysis, based on mathematical evaluations and stereological methods, has been used to study five left neonatal ovaries, removed from full-term neonates with a 46,XX karyotype free from malformations of the genital apparatus. Each ovary was completely cut obtaining serial sections and one 1-micron-thick section every 1,000 microns was examined. Ovarian length ranged from 9 to 17 mm (mean 13 mm), width from 3.5 to 7 mm (mean 5.7 mm), thickness from 2.5 to 5 mm (mean 4 mm), and volume from 82.23 to 198.3 mm3 (mean 125.88 mm3). In the ovarian cortex, primitive cortical tissue accounted for 10-20% of the total volume, follicles for 10-25% and interstitium for 35-45%; 10-30% of the organ consisted of inner medulla. The total follicle number ranged from 130,000 to 385,000 per ovary, with an average of 266,000 with 95% being represented by primordial follicles. In all ovaries examined follicular growth was still in process, with follicles at different stages of development.

Recent improvements in techniques in clinical assisted reproduction have led to an increased interest in the cryopreservation of human ovarian tissue as a way of preserving fertility and ovarian steroidogenic activity in young cancer patients. Acceptable follicular survival in frozen-thawed human ovarian tissue has generally been reported. Since a 0.3 mol/l sucrose concentration in cryopreservation solutions evidently increases human oocyte survival after cryopreservation, the aim of this study was to observe the effect of sucrose concentrations of 0.2 mol/l and 0.3 mol/l on human ovarian tissue survival after thawing. Ovarian cortical slices from 10 patients, 22-36 years of age, were cryopreserved slowly using 0.2 mol/l or 0.3 mol/l sucrose with 1,2-propanediol (1.5 mol/l) as the cryoprotectants. Light and electron microscopy were used for the histological analyses. Results showed that both treatments produced an increase in damaged cells; however, the use of 0.3 mol/l sucrose showed a smaller percentage of damaged germ cells than 0.2 mol/l sucrose, and therefore was less detrimental to the thawed ovarian tissue. However as the damage occurred principally in the stroma and follicular cells rather than in the oocytes, the suitability of these cryopreservation protocols must be further evaluated prior to considering the use of stored ovarian cortex for autografting after thawing.

The volumetric composition of the human ovary during the compartmentalization stage has been investigated using current stereological methods. Eight left ovaries removed from three fetuses (developmental age 20–25 weeks), four neonates, and one 8-month-old child all with a 46, XX karyotype, free from malformations of the genital apparatus, were completely cut obtaining serial sections and one 1 μm-thick section every 1,000 μm was examined. Ovarian volume was 30 mm3 at the 20th week of development, 36 mm3 at the 25th week, 129 mm3 at birth, and 287 mm3 at the eighth postnatal month. The primitive cortical tissue was the largest component of the fetal ovaries (17 mm3, corresponding to 60.2% of the organ). The second component was the interstitium (21% of the organ), followed by the medulla (11.8% of the organ). The primordial follicles occupied a small part of the organs: 1.8 mm3 at 20 weeks and 3.4 mm3 at 25 weeks (respectively 6.7% and 5.4% of the volumes of the relevant ovaries). At birth, most of the organ was composed of interstitial tissue (57 mm3, 44.2% of the volume) followed by the medulla (25 mm3, 20.3% of the volume). The germinal tissue occupied 46 mm3, mainly primitive cortical tissue (14.9% of the ovary) and primordial follicles (16.3% of the ovary), with a minor contribution from the antral follicles (about 3% of the ovary). At 8 months, the somatic tissue formed the majority of the organ (143 mm3 of stroma, corresponding to about 50% of the volume, and 43 mm3 of medulla, about 15% of the volume); the germinal tissue occupied about 101 mm3: most of this volume was given by the antral follicles (28.6% of the ovarian volume). © 1993 Wiley-Liss, Inc.