Natalia Rozwadowska

Changes in sub-cellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development.

BackgroundPreimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8–16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6.ResultsAt the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8–16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes.ConclusionsCell fate specification in cattle become evident earlier than presently anticipated – around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis

OBJECTIVE To identify potential biomarkers of azoospermia to determine a particular stage of spermatogenetic differentiation. DESIGN GeneChip Human Gene 1.0 ST microarray with validation at mRNA and protein levels. SETTING Basic research laboratory. PATIENT(S) Men with various types of nonobstructive azoospermia (n = 18) and with normal spermatogenesis (n = 4). INTERVENTION(S) Obtaining 31 testicular biopsy samples. MAIN OUTCOME MEASURE(S) Gene expression analysis using the Affymetrix Human Gene 1.0 ST microarrays on 14 selected genes according to the highest fold change, verified with quantitative polymerase chain reaction and on independent set of microarray samples. Western blot and immunohistochemistry were additionally performed. RESULT(S) The comparative analysis of gene expression profiles in the infertile and control groups resulted in the selection of 4,946 differentially expressed genes. AKAP4, UBQLN3, CAPN11, GGN, SPACA4, SPATA3, and FAM71F1 were the most significantly down-regulated genes in infertile patients. Global analysis also led to identification of up-regulated genes-WBSCR28, ADCY10, TMEM225, SPATS1, FSCN3, GTSF1L, and GSG1-in men with late maturation arrest. Moreover, the results from quantitative polymerase chain reaction and Western blot largely confirmed the microarray data. CONCLUSION(S) The set of selected genes can be used to create a molecular diagnostic tool to determine the degree of spermatogenic impairment for men with idiopathic nonobstructive azoospermia.

Cell-based therapy for heart failure: skeletal myoblasts.

Satellite cells are committed precursor cells residing in the skeletal muscle. These cells provide an almost unlimited regeneration potential to the muscle, contrary to the heart, which, although proved to contain cardiac stem cells, possesses a very limited ability for self-renewal. The idea that myoblasts (satellite cell progenies) may repopulate postinfarction scar occurred around the mid-1990s. Encouraging results of preclinical studies triggered extensive research, which led to the onset of clinical trials. These trials have shown that autologous skeletal myoblast transplantation to cure heart failure is feasible and relatively safe (observed incidences of arrhythmia). Because most of the initial studies on myoblast application into postischemic heart have been carried out as an adjunct to routine surgical procedures, the true clinical outcome of such therapy in regard to cell implantation is blurred and requires to be elucidated. The mechanism by which implantation of skeletal myoblast may improve heart function is not clear, especially in the light of inability of these cells to couple electromechanically with a host myocardium. Successful myoblast therapy depends on a number of factors, including: delivery to the target tissue, long-term survival, efficacious engraftment, differentiation into cardiomyocytes, and integration into the new, unique microenvironment. All these steps constitute a potential goal for cell manipulation aiming to improve the overall outcome of such therapy. Precise understanding of the mechanism by which cells improve cardiac function is essential in giving the sensible direction of further research.

ORIGINAL ARTICLE: The Role of IL-6, IL-10, TNF-α and its Receptors TNFR1 and TNFR2 in the Local Regulatory System of Normal and Impaired Human Spermatogenesis

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

The role of IL-6, IL-10, TNF-alpha and its receptors TNFR1 and TNFR2 in the local regulatory system of normal and impaired human spermatogenesis.

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

PRAME expression in head and neck cancer correlates with markers of poor prognosis and might help in selecting candidates for retinoid chemoprevention in pre-malignant lesions

OBJECTIVES PRAME (Preferentially Expressed Antigen in Melanoma) is a tumor-associated antigen recognized by immunocytes, and it induces cytotoxic T cell-mediated responses in melanoma. PRAME expression in tumors interferes with retinoic acid receptor (RAR) signaling thus promoting tumor progression. Here, we study PRAME expression in head and neck squamous cell carcinoma (HNSCC) to determine its potential clinical significance. MATERIALS AND METHODS PRAME expression in HNSCC was evaluated by immunohistochemistry in tissue microarrays of primary tumors (n=53), metastatic lymph nodes (n=8) and normal oral mucosa (n=11). Biopsies of dysplastic oral lesions (n=12) were also examined. PRAME expression levels in tissues were correlated with markers of poor prognosis in HNSCC. PRAME mRNA in HNSCC cell lines and in normal immortalized human keratinocytes (HaCaT cell line) was measured by qRT-PCR, and the protein expression by flow cytometry and western blots. RESULTS PRAME was expressed in HNSCC cell lines and HNSCC lesions. PRAME expression in dysplastic mucosa was variable. No or only weak expression was found in normal cells or tissues. PRAME expression levels significantly correlated with the tumor grade, size, nodal involvement and the clinical status of HNSCC patients. CONCLUSIONS Elevated PRAME expression associates with clinicopathologic markers of poor outcome in HNSCC and might identify potential candidates with pre-cancerous lesions for chemoprevention with retinoids.

Interleukin-1 system in testis — quantitative analysis

There is a growing body of evidence suggesting that interleukin-1 and other cytokines may influence mammalian testis function. IL-la activity seems to be tissue specific, and it appears in literature as a testicular interleukin-1 (tIL-1)1. Its activity was detected both in testicular interstitium and seminiferous epithelium. Being secreted in para-or autocrine manner, IL-1 inhibits steroidogenesis in Leydig cells, what suggests the modulatory role of this cytokine in LH-dependent testosterone synthesis. Sertoli cells are also capable to produce IL-1. The level of interleukin-1 expression in these cells is distinctly connected with the intensity of DNA synthesis in gametogenic cells. Thus IL-1 could be a nutritional factor, important for activity of seminiferous epithelium. Its expression both in Sertoli and Leydig cells depends on the local concentrations of hCG, FSH and LH and the other cytokines (e.g. TNFa) or growth factors in testis. Hence, IL-1 appears as another element of regulatory network essential for maintenance of testis physiology.2

Characterisation of Nuclear Architectural Alterations during In Vitro Differentiation of Human Stem Cells of Myogenic Origin

Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

Successful implantation of autologous muscle-derived stem cells in treatment of faecal incontinence due to external sphincter rupture

Dear Editor:The most common pathological mechanism of faecalincontinence is the insufficiency of the external anal sphinc-ter (EAS) caused by neurological or myogenic dysfunction.The myogenic mechanism of EAS insufficiency is usuallydue to direct mechanical damage during childbirth, traumaor surgery in anorectal region, whereas neurologicalaetiology involves either spinal or peripheral nerves disrup-tion—in most cases the pudendal nerve. Unfortunately, co-incidence of sphincter rupture with damage to pudendalnerves is quite common.Each skeletal muscle, including EAS, has the ability toregenerate to some degree and repair sustained damage. Inresponse to injury and/or muscle damage, so-called satellitecells are activated and become myoblasts—capable of in-tense proliferation. Myoblasts then differentiate and fusetogether to form new muscle fibres and connect withexisting ones, adding new portions of contractile tissue toexisting motoric units [1].Attempts of autotransplantation of myoblasts into dam-aged skeletal muscle were already made in animal models ofmuscular dystrophy, post-infarction myocardial dysfunctionand urethral sphincter insufficiency [2]. The results showedthat the transplanted myoblasts differentiate into musclefibres, connect with host motoric units, increase the amountof contractile elements in the muscle and improve its con-tractile activity. In 2001, Menasche et al. first transplantedautologous myoblasts into the post-infarction myocardialscar in human patients with cardiac failure, with significantimprovements in contractile function and clinical condition[3]. In Poland, the method of treating post-infarction heartfailure was performed for the first time a year later, withsimilar results [4].Basedonthose encouraging results,a pioneerexperimen-tal study was designed in attempt to enhance the function ofexternal anal sphincter using injections of autologousmuscle-derived stem cells. The study is designed as a pro-spective experimental study. It is being conducted by twocooperating research centres—the 3rd Department of Gen-eral Surgery, Jagiellonian University in Cracow and theDepartment of Reproductive Biology and Stem Cells, Insti-tute of Human Genetics, Polish Academy of Sciences inPoznan. We would like to present a case of the representa-tive patient enrolled to our study.A 20-year old male withfaecal incontinenceduetoanoldexternal anal sphincter rupture in a road accident was en-rolled to the study. Sphincter rupture had been repairedsurgically right after the accident (with an end-to-endsphincteroplasty). The patient underwent 6 months of bio-feedback training after the wounds were healed. At the timeof enrolment, he still complained of gas and loose stoolincontinence, daily soiling, with necessity to wear pads.Endoanal ultrasound showed a 8–10-mm scar on the leftcircumference of internal and external sphincter muscle,where anal canal was ruptured during the accident, andsurgically repaired afterwards. Anorectal manometryshowed decreased both mean resting and maximum squeezepressure, with short high pressure zone length. Endoanal

SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis

The transcription levels of stem cell factor (SCF) and c‐kit were examined using real‐time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c‐kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c‐kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c‐kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c‐kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c‐kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c‐kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.