Biomedical subjects

F Sinowatz

Publications and source records attributed to F Sinowatz.

At least 19 recordsLinked to original sources

PubMed2006

Cyclin D1 protein expression in human thyroid gland and thyroid cancer.

Cell cycle progression is facilitated by cyclin dependent kinases (CDKs) that are activated by cyclins, including Cyclin D1 and inhibited by CDK inhibitors. Evidence of the involvement of cyclin gene alterations and over expression of various cyclins in human cancer is growing. The role of Cyclin D1 in malignant progression of papillary carcinomas of the thyroid has yet to be established. We therefore studied the expression of Cyclin D1 protein in thyroid carcinomas of young Kuwaiti patients (36 cases of conventional papillary thyroid carcinoma, 12 cases of its follicular variant, one case of tall cell thyroid carcinoma and one case of medullary carcinoma) using immunohistochemistry. In 23 patients (46%) circumscribed areas of cells were detected that showed a distinct to strong nuclear staining for immunoreactive Cyclin D1 whereas the remaining bulk of the carcinoma cells were negative or only showed a slight cytoplasmic staining. None of the tested clinical or path histological parameters showed a statistically significant correlation with the focal immunostaining. This does not rule out that the detected foci with positive nuclear Cyclin D1 immunostaining are areas where a progressive transformation to a more malignant phenotype occurs which eventually leading to lymph node and distant metastases.

Adenocarcinoma, Follicular↗

PubMed2006

Localization of fibroblast growth factor I (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis, lactation and involution.

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.

Animals↗

PubMed2006

Immunohistochemical detection of macrophage migration inhibitory factor in fetal and adult bovine epididymis: release by the apocrine secretion mode?

Originally defined as a lymphokine inhibiting the random migration of macrophages, the macrophage migration inhibitory factor (MIF) is an important mediator of the host response to infection. Beyond its function as a classical cytokine, MIF is currently portrayed as a multifunctional protein with growth-regulating properties present in organ systems beyond immune cells. In previous studies, we detected substantial amounts of MIF in the rat epididymis and epididymal spermatozoa, where it appears to play a role during post-testicular sperm maturation and the acquisition of fertilization ability. To explore its presence in other species not yet examined in this respect, we extended the range of studies to the bull. Using a polyclonal antibody raised against MIF purified from bovine eye lenses, we detected MIF in the epithelium of the adult bovine epididymis with the basal cells representing a prominently stained cell type. A distinct accumulation of MIF at the apical cell pole of the epithelial cells and in membranous vesicles localized in the lumen of the epididymal duct was obvious. In the fetal bovine epididymis, we also detected MIF in the epithelium, whereas MIF accumulation was evident at the apical cell surface and in apical protrusions. By immunoelectron microscopy of the adult bovine epididymis, we localized MIF in apical protrusions of the epithelial cells and in luminal membrane-bound vesicles that were found in close proximity to sperm cells. Although the precise origin of the MIF-containing vesicles remains to be delineated, our morphological observations support the hypothesis that they become detached from the apical surface of the epididymal epithelial cells. Additionally, an association of MIF with the outer dense fibers of luminal spermatozoa was demonstrated. Data obtained in this study suggest MIF release by an apocrine secretion mode in the bovine epididymis. Furthermore, MIF localized in the basal cells of the epithelium and in the connective tissue could be responsible for regulating the migration of macrophages in order to avoid contact of immune cells with spermatozoa that carry a wide range of potent antigens.

Animals↗

PubMed2006

Region-specific expression of nitric oxide synthases in the bovine oviduct during the oestrous cycle and in vitro.

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17beta and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17beta. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.

Animals↗

PubMed2005

Expression and localization of growth factors and their receptors in the mammalian testis. Part I: Fibroblast growth factors and insulin-like growth factors.

It is now well established that normal development and function of testis are mediated by endocrine and paracrine pathways including hormones, growth factors and cytokines as well as by direct cell-to-cell contacts depending on tight, adhering and gap junctions. In the last two decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. Interestingly, most of these peptides are expressed not only in the adult mammalian testis during spermatogenesis but also during testicular morphogenesis in prenatal and postnatal life. Our study was launched to provide an overview of the expression, localization, and putative physiological roles of growth factors and their receptors in the mammalian testis. The growth factors considered in this part of our review are fibroblast growth factors and insulin-like growth factors. These factors are found in testicular cells in prenatal, postnatal, and adult animals and are implicated in the regulation of important testicular activities including testicular cord morphogenesis, modulation of testicular hormone secretion and control of spermatogenesis.

Animals↗

PubMed2005

The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.

Animals↗

PubMed2005

Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes.

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle--end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-beta signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

Animals↗

PubMed2004

Male and female effects on the in vitro production of bovine embryos.

A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3-93.4%) and blastocysts on day 7 (6.9-51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F-value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme.

Animals↗

PubMed2004

Clinicopathological features of extranodal lymphomas: Kuwait experience.

A total of 935 patients with extranodal non-Hodgkin lymphoma (NHL) diagnosed in the period between January 1985 and December 2000 in Kuwait Cancer Center, serving the whole population of Kuwait, were used to describe the clinicopathological and epidemiological features of extranodal lymphomas in Kuwait. Extranodal lymphomas accounted for 45% of all NHL observed during this time. All NHL cases from Kuwait Cancer registry were analyzed and pathologically reclassified using the latest WHO (2000) classification. The most common lymphoma observed was diffuse large B-cell lymphoma (58.60%) followed by Burkitt s lymphoma (BL) (3.80%). In the pediatric group, BL comprises more than two thirds of all patients (77.20%). The most common extranodal sites were stomach (19.70%) and skin (17.80%) in the adult group, large intestine (29.80%) and small intestine (19.30%) in the pediatric age group. The majority (73.40%) of adult extranodal lymphomas was in stage IE-IIE and had a very good prognosis. On the contrary, the majority of pediatric extranodal lymphomas were found to be in stage III and IV. Variations in treatment policies (single agent or combined chemotherapy, radiotherapy, combined modality treatment) adopted and changed during the time period of 16 years of this retrospective study were documented.

Adolescent↗

PubMed2004

Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography.

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

Animals↗

PubMed2003

Endocrine effects of environmental pollution on Xenopus laevis and Rana temporaria.

To determine the capacity of sewage treatment work effluents to disrupt the endocrine system under semifield conditions, two amphibian species, Xenopus laevis and Rana temporaria, were exposed to the effluent of a regional sewage treatment plant in South Bavaria during larval development until completion of metamorphosis. Exposure was carried out in river water (Würm) as a reference, and a 1:12-mixture sewage effluent representing the real situation on the spot, and in a higher concentration of sewage using a 1:2 mixture. An accidental impact of industrial wastewater into the reference and dilution medium, Würm, which was caused by a spate in the respective area during the sensitive period of sex differentiation of amphibian larvae, is assumed to be responsible for the relatively high percentage of females observed by histological analysis in all treatment groups. All of these values were higher than those determined in controls exposed to artificial tap water in laboratory experiments conducted in a comparable study design. Sex ratios between species, revealed by the semifield study with decreasing portions of females from control to 1:12 to 1:2, were strongly correlated. Determination of biomarker-mRNA-levels in Xenopus liver using semiquantitative RT-PCR at the end of the experimental phase, when exposure regime has turned into the initially expected situation with the highest load of potential estrogens in the effluent, followed by 1:2 and 1:12 mixture, resulted in a significant increase of Vitellogenin-mRNA in female juveniles exposed to the highest portion of sewage, whereas expression of both androgen and estrogen receptor-mRNA showed no clear differences. The results concerning the induction of estrogenic biomarkers are in accordance with our findings for estrogen receptor binding of sample extracts from the Würm and sewage taken in parallel at the end of the experiment, when sewage extracts possessed a much higher ability to displace [3H]estradiol from the estrogen receptor than the ones extracted from the mixtures.

Animals↗

PubMed2003

Morphology of canine cumulus-oocyte complexes in pre-pubertal bitches.

The morphology of canine cumulus-oocyte complexes (COCs) before puberty is still unknown. Therefore, the aim of our study was to elucidate the morphological characteristics of pre-pubertal oocytes and cumulus cells by light microscopy, scanning electron microscopy and transmission electron microscopy. The pre-pubertal oocyte was characterized by accumulation of lipid yolk droplets in the cytoplasm as well as high energy metabolism, low protein synthesis and high transcriptional activity of the cumulus cells. The cumulus cells, which revealed a prominent nucleus and few cytoplasm, communicated with each other by few short processes and exhibited merely a small amount of processes reaching the oocyte. Our studies imply that both the oocyte and the cumulus cells of canine COCs before puberty reveal characteristic morphological features which are correlated with changes in oocyte metabolism and cumulus cell communication.

Animals↗

PubMed2003

On the species specificity of sperm binding and sperm penetration of the zona pellucida.

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species-specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co-incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.

Acrosome↗

PubMed2003

Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

Animals↗

PubMed2003

Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution.

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

Animals↗

PubMed2002

Expression of cytokeratin 18 during pre- and post-natal porcine lung development.

The expression pattern of the intermediate filament protein cytokeratin 18 (CK 18) is described during pre- and post-natal development of the porcine lung using a monoclonal antibody against human CK 18. Lungs from 16 foetuses in pseudoglandular, canalicular, saccular and alveolar stages of lung development and lungs from 12 pigs ranging in age from birth to 49 days after birth were studied by immunohistochemistry. In the early pseudoglandular stage of development (day 70 of gestation) all the columnar epithelial cells lining the tubular endbuds strongly expressed CK 18 predominantly in the apical cell compartment. A modest staining was found in the more cuboidal cells of the canalicular stage (day 80 of gestation) where the labelling occurred as a distinct positive rim at the apical cell membrane in most of the cells lining the canaliculi. In 96- and 100-day-old foetuses, parts of the gas exchanging area were formed as terminal sacs by extreme attenuation of the epithelium. In this stage, CK 18 was clearly detectable in the flat type I as well as in the cuboidal type II alveolar epithelial cells. A marked change of the CK 18 expression pattern occurred during formation of the alveoli by septal outgrowth and maturation of the epithelium in 105- and 111-day-old foetuses. Differentiated type I cells no longer expressed CK 18, whereas type II cells were still labelled. Moreover, a specific change in the subcellular distribution pattern from the luminal periphery in immature porcine type II cells to a cytoplasmic localization in differentiated type II cells could be observed. Our investigation additionally demonstrated that the epithelium of bronchi, bronchioli and terminal bronchioli expressed CK 18 in all pre- and post-natal developmental stages. From the 96 days of gestation onwards the epithelial cells of developing bronchial glands were also labelled. Our results clearly show that during porcine lung development profound changes in the cellular expression pattern of CK 18 occur and that CK 18 can be regarded as a selective marker for differentiated porcine alveolar type II cells from the 105th day of gestation onwards. We also assume that the intermediate filament CK 18 could be of significance in the maturation process of the type II alveolar cells.

Age Factors↗

PubMed2002

Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro.

Hyaluronic acid (HA) is the main glycosaminoglycan present in follicular, oviductal and uterine fluids. The main functions of HA include dynamic processes that are mediated through interaction with extracellular matrix components, regulation of gene expression, cell proliferation and cell differentiation. HA increases the viscosity of solutions and also has several physiological functions, including regulation of water distribution and water-binding capacity. The addition of 6 mg HA ml(-1) to synthetic oviduct fluid (SOF; SOF-HA) culture medium on day 5 (IVF = day 0) significantly (P < 0.001) increased the viscosity of the medium in comparison with SOF culture medium containing BSA (SOF-BSA). On day 8, rate of blastocyst development in SOF-HA culture medium was significantly (P < 0.05) higher than in SOF-BSA culture medium (38.2 versus 29.3%). The number of trophectoderm cells and the total number of cells of expanded blastocysts cultured in the presence of HA were significantly (P < 0.01) higher in comparison with expanded blastocysts cultured in the presence of BSA (88.9 +/- 7.3 versus 67.6 +/- 3.0 and 130.1 +/- 10.9 versus 104.8 +/- 2.5, respectively). After freezing and thawing, the percentage of day 8 embryos that re-expanded and hatched when cultured with SOF-HA was greater than that of embryos cultured with SOF-BSA (11.3 and 10.5% versus 75.5 and 36.8%, respectively). After thawing, the ATP contents of in vivo-derived, SOF-HA and SOF-BSA expanded blastocysts were similar. The embryos cultured with HA showed less ultrastructural deviation and de-differentiation after freezing and thawing than the embryos cultured with BSA. This study demonstrates that HA improves the developmental capacity of bovine embryos under in vitro conditions and is warranted as a supplement for in vitro production of bovine embryos, particularly if they are to be cryopreserved.

Adenosine Triphosphate↗

PubMed2002

Effects of a novel co-culture system on development, metabolism and gene expression of bovine embryos produced in vitro.

An in vitro model using co-culture of bovine in vitro-produced (IVP) embryos and bovine oviduct epithelial cells (bOECs) was established to study embryo-maternal interactions in the oviductal environment. In vitro conditions maintaining differentiated growth of oviductal cells were determined by evaluating several media supplemented with different sera at various concentrations. Morphological features were used as indicators of physiological growth, and it became obvious that synthetic oviduct fluid (SOF) supplemented with either oestrous cow serum (OCS) or dextran-coated charcoal-treated fetal calf serum (DCC-FCS) helped to prevent dedifferentiation of bOECs (Expt 1). RT-real-time-PCR analysis revealed an increased mRNA content of the oviduct-specific glycoprotein GP 85-97 when using lower serum concentrations (2 and 5% compared with 10%; Expt 2). In subsequent experiments in which cell-free cultured controls and co-cultured embryos were compared, co-cultured embryos showed an increased rate of cleavage (P < 0.05) after 3 days. Successive cell-free culture until day 8 resulted in a lower rate of blastocyst development (P < 0.05) and reduced ATP content (P < 0.05) of co-cultured versus control embryos (Expt 3). Long-term co-culture (8 days) in SOF with 5% OCS increased the expression of developmentally relevant genes (glucose transporter 1 (Glut-1) and heat shock protein (HSP 70)) in co-cultured versus control embryos (Expt 4). Higher embryonic Glut-1 mRNA expression after co-culture was obvious when using 10% DCC-FCS, but was not significant when culture medium was supplemented with 10% rather than 5% OCS (Expt 5). In conclusion, SOF with 5% OCS supports differentiated growth of bOECs. Co-culture under these conditions improves early cleavage rate, but not blastocyst development, and increases the expression of developmentally relevant genes influenced by type of serum and serum concentration.

Animals↗