Cells Of Different Developmental Stages Research Articles

Biological Parameters and Spermatogenesis in Razorfish (Pelecus cultratus) Population Inhabiting the Largest Shallow Lake of Central Europe (Lake Balaton): Studies for In Vitro Conservation Purposes

The study aimed to investigate body parameters and the process of spermatogenesis from April to September. In addition, it sought to test the applicability of sperm cryopreservation for conservation purposes in a razorfish (Pelecus cultratus) population of Lake Balaton, the largest shallow lake in Central Europe. During the aforementioned period, measurements were taken for the standard length (SL, cm) and body weight (BW, g), and the sex of specimens was determined. Cells at different stages of spermatogenesis (spermatogonia-SG, spermatocytes-SC, spermatids-ST, and spermatozoa-SZ) were quantified monthly for each male sample. Sperm samples collected at the end of May were cryopreserved using a method designed for common carp. No significant differences were found between males (SL: 25 ± 2 cm and BW: 146 ± 38 g) and females (SL: 26 ± 3 cm and BW: 168 ± 53 g) in terms of measured body parameters. No significant correlations were found between the sex, SL, and BW. High standard deviations were observed for all mean values in all sampling periods, possibly due to the low number of individual samples per month. A significantly higher proportion of SC compared to SZ was found in April. In May, no differences were observed between the four groups. Significantly more SG and SZ than SC and ST were observed in June. In August and September, a slight dominance in the number of SG was recorded, with no differences measured among the cells in different developmental stages. The males studied exhibited a low gonadosomatic index (0.92% ± 0.27%). A significant reduction was recorded in motility (MOT), progressive motility (pMOT), and in most of the kinetic parameters (distance curved line-DCL, curvilinear velocity-VCL, straight line velocity-VSL, and beat cross frequency-BCF). The spermiation of males could have started in May and conceivably lasted until the end of June. It is recommended to increase sperm quality and quantity before cryopreservation.

Topological Data Analysis in Time Series: Temporal Filtration and Application to Single-Cell Genomics

The absence of a conventional association between the cell–cell cohabitation and its emergent dynamics into cliques during development has hindered our understanding of how cell populations proliferate, differentiate, and compete (i.e., the cell ecology). With the recent advancement of single-cell RNA sequencing (RNA-seq), we can potentially describe such a link by constructing network graphs that characterize the similarity of the gene expression profiles of the cell-specific transcriptional programs and analyze these graphs systematically using the summary statistics given by the algebraic topology. We propose single-cell topological simplicial analysis (scTSA). Applying this approach to the single-cell gene expression profiles from local networks of cells in different developmental stages with different outcomes reveals a previously unseen topology of cellular ecology. These networks contain an abundance of cliques of single-cell profiles bound into cavities that guide the emergence of more complicated habitation forms. We visualize these ecological patterns with topological simplicial architectures of these networks, compared with the null models. Benchmarked on the single-cell RNA-seq data of zebrafish embryogenesis spanning 38,731 cells, 25 cell types, and 12 time steps, our approach highlights gastrulation as the most critical stage, consistent with the consensus in developmental biology. As a nonlinear, model-independent, and unsupervised framework, our approach can also be applied to tracing multi-scale cell lineage, identifying critical stages, or creating pseudo-time series.

Proinflammatory cytokines induce rapid, NO-independent apoptosis, expression of chemotactic mediators and interleukin-32 secretion in human pluripotent stem cell-derived beta cells

Aims/hypothesisThe aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells.MethodshPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-βH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1β, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways.ResultsA 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-βH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including IL-32, CXCL9 and CXCL10 in SC-beta cells and in non-insulin-producing cells.Conclusions/interpretationOur results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes.Graphical abstract

N6-Methyladenosine detected in RNA of testicular germ cell tumors is controlled by METTL3, ALKBH5, YTHDC1/F1/F2, and HNRNPC as writers, erasers, and readers.

Type II testicular germ cell tumors (GCTs) arise from a common precursor lesion (germ cell neoplasia insitu) and are stratified into seminomas and non-seminomas, which differ considerably in morphology, gene expression, and epigenetic landscape. The N6-methyladenosine (6mA) epigenetic modification is the most abundant modification in mRNA and is also detectable in eukaryotic DNA. The functional role of 6mA is not fully understood, but 6mA residues may influence transcription by affecting splicing, miRNA processing, and mRNA stability. Additionally, the methyl group of 6mA destabilizes Watson-Crick base-pairing affecting RNA structure and protein binding. Here, we analyzed the presence of the 6mA epigenetic modification in germ cells and GCT tissues and cell lines. We screened for the presence of 6mA in DNA and RNA by immunohistochemistry, mass spectrometry or ELISA-based quantification assays. Additionally, expression of 6mA writer-, eraser- and reader-factors was analyzed by microarrays, qRT-PCR, western blotting and screening of public databases. We demonstrate that 6mA is detectable in RNA, but not DNA, of GCT cell lines and tissues, fibroblasts, and Sertoli cells as well as germ cells of different developmental stages. Based on expression analyses, our results suggest METTL3, ALKBH5, YTHDC1, YTHDF1, YTHDF2 and HNRNPC as main writers, erasers, and readers of the 6mA modification in GCTs. Owing to the lack of 6mA in DNA of GCTs, a functional role in regulating DNA transcription can be excluded. Interestingly, expression levels of 6mA regulators are comparable between tumor and normal tissues/cells, suggesting a similar mechanism of 6mA regulation in RNA. Finally, we demonstrate that 6mA levels in RNA increase upon differentiation of GCT cell lines, suggesting a role of 6mA in cell fate decisions. In summary, our data provide the starting point for further experiments deciphering the role of 6mA in the RNA of GCTs.

Spermatogonial stem cells differentiation and testicular lobules formation in a seasonal breeding teleost: The evidence from the heat-induced masculinization of genetically female Japanese flounder (Paralichthys olivaceus)

Germ cells undergo a series of cellular changes including differentiation, mitosis, meiosis and maturation, and eventually develop into a large number of functional gametes. The available data regarding teleost gametogenesis in seasonal batch spawners is limited. In this study, we investigated spermatogenesis with special attention on spermatogonia differentiation using heat-induced masculine juveniles of genetically female Japanese flounder. Meanwhile, the Nanos2 expression had been detected by immunohistochemistry for analysis of spermatogonial stem cells (SSCs) distribution. Spermatogonia began mitosis at 35 dph, and basement membrane firstly appeared and gradually surrounded and separated the spermatogonia (type A) into single and paired status. At this period, the spermatogonia continuously maintained mitotic proliferation. As a result, the number of spermatogonia including isolated and clusters (2–8 spermatogonia) significantly increased in the presumptive testes. From 85 dph to 120 dph, with the mitosis of spermatogonia, germline acinar-clusters formed. In the clusters, type A spermatogonia differentiated into type B, and multi-spermatogonia surrounded by several sertoli cell formed cysts，which represented the formation of lobular precursors. After that, type B spermatogonia began meiosis, which indicated the initiation of spermatogenesis. In adult testes, most type A spermatogonia distributed in the peripheral region and a few clung to a basement membrane in the internal germinal epithelium. Various spermatogenic cysts with germ cells in different developmental stages existed in a testicular lobules, moreover the germ cells in earlier stages resided in the distal termini, and the advanced stages were adjacent to the spermatic duct of testes. Therefore, the testes of Japanese flounder belonged to an intermediated distribution of SSCs, which might contribute greatly to multiple spermiation during breeding season. These findings would improve the understanding the mechanisms of SSCs differentiation and testicular development, and may be of great value in future studies of the spermatogenesis regulation.

Abstract 4993: Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade

Abstract Breast cancers are classified into five intrinsic subtypes based on gene expression profile. It is suggested that these intrinsic subtypes originate from specific developmental stage of breast epithelial cell hierarchy, stem-progenitor-mature cell. However, normal breast epithelial cell lines representing these intrinsic subtypes are yet to be created. Using normal breast tissues of ancestry-mapped Caucasian, African American, and Hispanic women and a primary cell culturing system that allows growth of normal epithelial cells of different developmental stages including estrogen receptor-positive mature luminal cells, we created 15 human telomerase-immortalized breast epithelial cell lines. These cells formed acini on a matrigel and ductal structures on 3-dimensional collagen or hydrogel, indicating that these cell lines have retained characteristics of normal breast epithelial cells. RNA sequencing and PAM50 intrinsic subtype clustering algorithms were used to identify the intrinsic subtypes of the immortalized cell lines together with two well characterized “normal” breast epithelial cell lines MCF10A and HMEC as well as luminal breast cancer cell line MCF-7. Unlike MCF10A and HMEC, which are enriched for basal-like gene expression pattern, our cell lines classified into luminal A, basal, and normal-like subtypes. This was also reflected in the immunofluorescence staining with basal marker KRT14 and luminal marker KRT19. Few of these cell lines were dual positive for KRT14 and KRT19, but in varying proportions. Cell lines representing claudin-low subtypes were also created, which are phenotypically (CD201+/EpCAM-) different from the above cell lines. Cell lines showed inter-individual differences in stemness/differentiation capabilities and variable basal activity of signaling molecules such as NF-kB, AP-1 and pERK, which is consistent with, possibly reflecting, recent discoveries of genetic variations in gene regulatory regions among general population that contribute to widespread differences in gene expression/signaling under “normal” state. As majority of breast cancers are believed to originate from luminal progenitor cells, which are well represented our cell lines, these cell lines are ideal to delineate the impact of inter-individual and ethnic differences in normal breast biology on breast cancer initiation and progression as well as to determine whether cell-type-origin instead of genomic aberration drives intrinsic subtype-enriched gene expression patterns in breast tumors. Citation Format: Brijesh Kumar, Mayuri Prasad, Manjushree Anjanappa, Poornima Nakshatri, Natascia Marino, Anna Maria Storniolo, Xi Rao, Sheng Liu, Jun Wan, Yunlong Liu, Harikrishna Nakshatri. Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4993.

Multiscale 3D view of the genome

Ultradeep sequencing of Hi-C data from mouse cells in different developmental stages can address outstanding questions about 3D chromatin function.

Molecular genetic control of plant stomatal development

Stomata widely exist in the epidermis of plant shoot. They are the main passageway for gas exchange between plants and the environment. Stomata regulate many physiological processes, such as photosynthesis and transpiration. The protodermal cells undergo a series of stereotypical cell divisions and differentiation, and finally produce stomata. Several transcription factors control the initiation, proliferation and differentiation of stomatal cells in different developmental stages. The cell-cell communication, which is mediated by the ligand-receptor and MAPK cascade, ensures correct stoamtal patterning. A few polarity proteins have been reported to direct the orientation of asymmetric cell divisions. Additionally, plant hormones and environmental factors also influence stomatal development. All these factors together build the molecular genetic network of stomatal development. In this review, we summarize the recent advances of the network.

Abstract 8: Reprogramming of Adult Mammalian Cardiomyocytes Towards Proliferation and Regeneration Through Comparative Analyses of Epicardial and Myocardial Cells

Heart development and regeneration require an elegant balance of cell proliferation and differentiation. In both adult zebrafish and neonatal mouse cardiac regeneration, new cardiomyocytes are shown to originate from pre-existing cardiomyocytes through de-differentiation of mature cells to the immature progenitor cell state, with developmental gene program reactivation and cell cycle reentry. Interestingly, after adult mammalian cardiac injury, both cardiac muscle cells and the epicardial cells that envelope the heart reinitiate developmental gene programs. However, adult epicardial cells are able to proliferate following injury but adult cardiomyocytes are not capable. We investigated the genetic circuitry of mouse epicardial cells and myocardial cells in different developmental stages and in response to injury. Such comparative analyses revealed a group of ~50 candidate genes that may be responsible for the permanent cell cycle arrest of cardiomyocytes. We generated adenoviruses that express these candidate genes individually, and demonstrated that the mixed viral pool possessed a robust activity to promote proliferation of adult mouse cardiomyocytes. Our comparative approach and functional screens may lead to identification of the dormant genetic circuitry in adult mammalian heart that can be reactivated to drive robust cardiomyocyte proliferation and regeneration.

Spermatogenesis and related plasma androgen and progestin level in wild male golden mahseer, Tor putitora (Hamilton, 1822), during the spawning season.

Testicular development and plasma levels of sex steroid [11-ketotestosterone (11-KT), testosterone (T) and 17,20β-dihydoxy-4-pregnen-3-one (17,20β-P)] were studied for the first time in wild golden mahseer, Tor putitora. Testicular development was investigated by macroscopic observation and histology of the gonads, whereas steroids were measured by enzyme-linked immunosorbent assay. Based on macroscopic observation and germ cell types present in gonad histology, the testes of T. putitora were divided into five developmental stages: immature [stage I; spermatogonia (SPG)], early spermatogenesis [stage II; SPG and spermatocytes (SPC)], late spermatogenesis [stage III; SPG, SPC, spermatids (SPD) and spermatozoa (SPZ)], spermiation (stage IV; SPZ) and post-spawning (stage V; SPG, SPD and SPZ). During the stage I of the testes, the lowest levels of plasma sex steroid and gonadosomatic index (I G) were recorded. The highest plasma level of T was 0.89±0.09ng/mL and 11-KT was 4.23±0.54ng/mL, which was during the stage III and IV, respectively. The peak in 11-KT was coincident with the peak in I G (1.65±0.12%). The lowest T and 11-KT levels were 0.25±0.02ng/mL and 0.47±0.09ng/mL, respectively, which was at stage I. Plasma levels of 17,20β-P increased significantly at stage III (1.04±0.06ng/mL) and stage IV testes (1.28±0.03ng/mL) and then declined in post-spawned fish. This indicates that 17,20β-P could also be a possible maturation-inducing steroid in this fish. The condition factor (K) significantly decreased during the testicular development and was lowest at spermiation stage (0.53±0.02%). The proportion of running male peaked concomitantly with the appearance of stage IV testes. Presence of germ cells of different developmental stages indicates that T. putitora male is a multiple spawner, and the information generated here is important for developing a captive breeding, culture and conservation programs for this endangered coldwater Himalayan fish species.

Different expression profiles of micro-RNA in epidermal cells of different developmental stages

Objective To investigate the difference in expression profiles of micro-RNA (miR-NA) between human epidermal stem cells and epidermal keratinocytes.Methods (1) Human primary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type Ⅳ collagen coated chosen method.Growth of cells cultured in vitro was observed by inverted microscope.Monoclonal antibody of integrinβ1,keratin 1 (CK1),CK10,and CK19 were detected by immunocytochemical staining.(2)Total RNA was respectively isolated from epidermal stem cells and epidermal keratinocytes by Trizol-based single-step procedure,detected by formaldehyde denaturing gel electrophoresis,purified by mirVanaTM miRNA isolation kit,and then labeled and hybridized by miRNA labeling and hybridization kit.Images of hybridization were analyzed using the Feature Extraction (Version 10.7).Data normalization and difference analysis were performed with GeneSpring (GX 10.0).Moreover,miRNA microarray results were confirmed by RT-PCR.(3) Target genes of differently expressed miRNA were predicted.Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinβ1 and CK19 were positive.Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive.(2) Of the epidermal stem cells,31 miRNAs were up-regulated and 153 down-regulated.Besides,significantly up-regulated miRNAs were hsa-miRNA-125b-3p,hsa-miRNA-197-5p,and hsa-miRNA-376a-3p,while significantly down-regulated miRNAs were hsa-miRNA-203,hsa-miRNA-29b-3p,and hsa-miRNA-34a-3p.Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR-29b-3p revealed high concordance with the microarray results.(3) Some miRNAs target genes indicated that miRNA related to cell proliferation,differentiation,apoptosis,aging and other biological characteristics.Conclusion Distinct differences in miRNA expression profiles are detected between human epidermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability. Key words: Epidermis; Stem cells; Gene expression profiling

X inactivation: a histone protects from reprogramming by the frog

Genes on the inactive X chromosome (Xi) of female mammals are repressed in a remarkably stable manner and reactivation of transcription is generally not observed unless the cell is reprogrammed to an early embryonic type. In this issue of The EMBO Journal , Pasque et al (2011) use a reprogramming system in frog oocytes to study the stability of Xi chromatin in cells of different developmental stages and identify the histone variant macroH2A as a factor preventing transcriptional activation.

Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development

Sensitivity to salinity varies between plant organs and between cells of different developmental stages within a single organ. The physiological and molecular bases for the differential responses are not known. Exposure of plants to salinity is known to induce formation of reactive oxygen species (ROS), which are involved in damage mechanisms but also in cell growth processes. The objective of this study was to elucidate developmental-stage-specific and organ-specific involvement of oxidative defense in the plant response to salinity in maize ( Zea mays L.). Plants were grown in nutrient solution containing 1 mM NaCl (control) or 80 mM NaCl. The oxidative stress response and damage symptoms along the cell developmental gradient in growing and mature tissue of leaves and roots were examined. Unlike leaves, roots did not suffer oxidative damage in either growing or mature cells and demonstrated reduced antioxidant response. This may reflect different requirements of ROS for growth mechanisms of leaf and root cells. In leaves, growing tissue demonstrated higher stimulation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity under salinity than mature tissue, whereas mature tissue demonstrated higher stimulation of catalase. These results indicate differential roles for these ROS-scavenging enzymes at different cell developmental stages. Because ROS are required for cell expansion, the higher increase in SOD and APX activities in the growing leaf cells that resulted in reduction of ROS content under salinity could lead to the inhibition of cell growth under salinity.

Direct hepatic differentiation of mouse embryonic stemcells induced by valproic acid and cytokines

To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

Spermatid Differentiation and Sperm Ultrastructure of the Granular Ark, Tegillarca granosa (Bivalvia: Arcidae)

This study describes spermatogenesis and sperm ultrastructure of the granular ark, Tegillarca granosa using light and electron microscopy. In the active spermatogenic season, the testis comprises many spermatogenic follicles that contain germ cells in different developmental stages. Primary spermatocytes in the pachytene stage are characterized by synaptonemal complexes. The early spermatids are characterized by the appearance of several Golgi bodies, increased karyoplasmic electron density, and tubular mitochondria. The mass of proacrosomal granules consists of numerous heterogeneous granules with high electron density that are about 20 nm in diameter. From the midstage of spermiogenesis, the well-developed mitochondria in the cytoplasm aggregate posterior to the nucleus and surround the proximal and distal centrioles. The proacrosomal granules condense and form a single acrosome with a thin envelope. During late spermiogenesis, the acrosome begins to elongate becoming conical. The sperm is approximately <TEX>$35.0{\mu}m$</TEX> long and consists of a head, midpiece, and tail. The head comprises a round nucleus and a conical acrosome. A micro fibrous axial rod is observed between the nucleus and acrosome. The midpiece has a calyx-like structure with five mitochondria, and the tail, which has the typical "9+2" microtubular system, originates from the distal centriole.

Comparative analysis of nuclear proteins of B cells in different developmental stages

The developmental stage-specific regulation of V(D)J recombination, a gene rearrangement process of antigen receptor gene segments, is tightly controlled in cells. Here we screened proteins uniquely or differentially expressed among three developmentally distinguishable B cells (pro-B, pre-B and mature B cells) using two-dimensional gel electrophoresis and mass spectrometry. Chromatin assembly factor 1 was uniquely expressed in pro-B cells. Purine nucleotide phosphorylase, LCK, E2A and many other unidentified proteins were dominantly present in the nucleus at the early stage of B cell development where the V(D)J recombination process occurs. Also, few proteins including guanidine nucleotide binding proteins were exclusively expressed in pre-B cell. Such findings can provide some information to help understand the developmental regulation of gene rearrangement occurring during B cell development.

Endometriosis--a stem cell disease?

Endometriosis is an estrogen-dependent and chronic disease with an unknown etiology and pathogenesis. It is however likely and well accepted that retrograde menstruation of endometrial cells into the pelvic cavity is the origin of this disease in many cases. Here we discuss a model in which retrogradely menstruated endometrial cells have different inherent developmental properties because they represent in fact a mixture of different developmental cell stages. These stages can be distinguished in part by the expression of marker proteins such as cytokeratin (intermediate filament protein of epithelial cells) or E-cadherin (intercellular adhesion protein of epithelial cells and metastasis suppressor molecule). Cytokeratin-positive E-cadherin negative cells, for example, would be less differentiated epithelial cells than cytokeratin-positive E-cadherin positive cells. In analogy to findings in other cell systems we assume that the cells which are undifferentiated or not fully differentiated still have the potential to give rise to differentiated daughter cells and, on the other hand, could be maintained as a pool of rather undifferentiated cells and capable of self renewal. This feature would be similar to stem cells (SC) and cells with plasticity. Interestingly we find epithelial cells of different developmental stages in deep infiltrating (e. g. of colon) or peritoneal endometriotic lesions. Therefore we conclude that less differentiated cells in retrogradely menstruated endometrial cell populations possibly representing SC features or plasticity might be the cellular source of primary endometriotic lesions and those present in lesions may contribute to the persistence of the disease by detaching and forming secondary lesions.

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers.

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.

Expression of the tissue kallikrein-kinin cascade in granulosa cells of the ovary.

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin-treated and non-treated control rats. Recently, we demonstrated that tissue kininogenase showed intense immunolabeling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells associated with ovarian vascularization possess the same capacity to express the kallikrein-kinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stages and segments of the ovarian follicle by immunoperoxidase assay, confocal fluorescent microscopy and in situ hybridization.

Proliferation, cell death and reelin expression during development of the retina in a teleost, the common trout (Salmo trutta fario)

We have analysed the spatiotemporal pattern of cell proliferation and apoptosis during the development of the retina in a teleost. Distribution of proliferating cells in different developmental stages (embryos, alevins, and juveniles) was analysed with an antibody against proliferating cell nuclear antigen (PCNA). After establishment of the retinal lamination, PCNA-immunoreactive (PCNA-ir) cells were frequently observed in all cell layers, with a high majority of proliferating cells located at the marginal (growing) retina. Clusters of PCNA-ir cells were characteristic of the inner nuclear layer (INL). The distribution of apoptotic cells was studied by the TUNEL method, which has revealed significant cell death before completion of the retinal lamination. We have also used an antibody against Reelin (RELN) as a marker for retinal cells that secrete this glycoprotein to the extracellular space. The pattern of RELN expression, as well as that of proliferating and dying cells was very similar to that described in tetrapods, suggesting that this protein regulates the cell migration and synaptogenesis in the different developmental stages of the retina.